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  • Cyclin A/Cdk1 modulates Plk1 activity in prometaphase to regulate kinetochore-microtubule attachment stability
    eLife (IF 7.725) Pub Date : 2017-11-20
    Ana Maria G Dumitru, Scott F Rusin, Amber E M Clark, Arminja N Kettenbach, Duane A Compton

    The fidelity of chromosome segregation in mitosis is safeguarded by the precise regulation of kinetochore microtubule (k-MT) attachment stability. Previously, we demonstrated that Cyclin A/Cdk1 destabilizes k-MT attachments to promote faithful chromosome segregation. Here, we use quantitative phosphoproteomics to identify 156 Cyclin A/Cdk1 substrates in prometaphase. One Cyclin A/Cdk1 substrate is myosin phosphatase targeting subunit 1 (MYPT1), and we show that MYPT1 localization to kinetochores depends on Cyclin A/Cdk1 activity and that MYPT1 destabilizes k-MT attachments by negatively regulating Plk1 at kinetochores. Thus, Cyclin A/Cdk1 phosphorylation primes MYPT1 for Plk1 binding. Interestingly, priming of PBIP1 by Plk1 itself (self-priming) increased in MYPT1-depleted cells showing that MYPT1 provides a molecular link between the processes of Cdk1-dependent priming and self-priming of Plk1 substrates. These data demonstrate cross-regulation between Cyclin A/Cdk1-dependent and Plk1-dependent phosphorylation of substrates during mitosis to ensure efficient correction of k-MT attachment errors necessary for high mitotic fidelity.

    更新日期:2017-11-20
  • Ribosome surface properties may impose limits on the nature of the cytoplasmic proteome
    eLife (IF 7.725) Pub Date : 2017-11-20
    Paul E Schavemaker, Wojciech M Śmigiel, Bert Poolman

    Much of the molecular motion in the cytoplasm is diffusive, which possibly limits the tempo of processes. We studied the dependence of protein mobility on protein surface properties and ionic strength. We used surface-modified fluorescent proteins (FPs) and determined their translational diffusion coefficients (D) in the cytoplasm of Escherichia coli, Lactococcus lactis and Haloferax volcanii. We find that in E. coli D depends on the net charge and its distribution over the protein, with positive proteins diffusing up to 100-fold slower than negative ones. This effect is weaker in L. lactis and Hfx. volcanii due to electrostatic screening. The decrease in mobility is probably caused by interaction of positive FPs with ribosomes as shown in in vivo diffusion measurements and confirmed in vitro with purified ribosomes. Ribosome surface properties may thus limit the composition of the cytoplasmic proteome. This finding lays bare a paradox in the functioning of prokaryotic (endo)symbionts.

    更新日期:2017-11-20
  • Functional divergence of paralogous transcription factors supported the evolution of biomineralization in echinoderms
    eLife (IF 7.725) Pub Date : 2017-11-20
    Jian Ming Khor, Charles A Ettensohn

    Alx1 is a pivotal transcription factor in a gene regulatory network that controls skeletogenesis throughout the echinoderm phylum. We performed a structure-function analysis of sea urchin Alx1 using a rescue assay and identified a novel, conserved motif (Domain 2) essential for skeletogenic function. The paralogue of Alx1, Alx4, was not functionally interchangeable with Alx1, but insertion of Domain 2 conferred robust skeletogenic function on Alx4. We used cross-species expression experiments to show that Alx1 proteins from distantly related echinoderms are not interchangeable, although the sequence and function of Domain 2 are highly conserved. We also found that Domain 2 is subject to alternative splicing and provide evidence that this domain was originally gained through exon extension. Our findings show that a gene duplication event permitted the functional specialization of a transcription factor through changes in exon-intron organization and thereby supported the evolution of a major morphological novelty.

    更新日期:2017-11-20
  • Functional divergence of paralogous transcription factors supported the evolution of biomineralization in echinoderms
    eLife (IF 7.725) Pub Date : 2017-11-20
    Jian Ming Khor, Charles A Ettensohn

    Alx1 is a pivotal transcription factor in a gene regulatory network that controls skeletogenesis throughout the echinoderm phylum. We performed a structure-function analysis of sea urchin Alx1 using a rescue assay and identified a novel, conserved motif (Domain 2) essential for skeletogenic function. The paralogue of Alx1, Alx4, was not functionally interchangeable with Alx1, but insertion of Domain 2 conferred robust skeletogenic function on Alx4. We used cross-species expression experiments to show that Alx1 proteins from distantly related echinoderms are not interchangeable, although the sequence and function of Domain 2 are highly conserved. We also found that Domain 2 is subject to alternative splicing and provide evidence that this domain was originally gained through exon extension. Our findings show that a gene duplication event permitted the functional specialization of a transcription factor through changes in exon-intron organization and thereby supported the evolution of a major morphological novelty.

    更新日期:2017-11-20
  • Cyclin A/Cdk1 modulates Plk1 activity in prometaphase to regulate kinetochore-microtubule attachment stability
    eLife (IF 7.725) Pub Date : 2017-11-20
    Ana Maria G Dumitru, Scott F Rusin, Amber E M Clark, Arminja N Kettenbach, Duane A Compton

    The fidelity of chromosome segregation in mitosis is safeguarded by the precise regulation of kinetochore microtubule (k-MT) attachment stability. Previously, we demonstrated that Cyclin A/Cdk1 destabilizes k-MT attachments to promote faithful chromosome segregation. Here, we use quantitative phosphoproteomics to identify 156 Cyclin A/Cdk1 substrates in prometaphase. One Cyclin A/Cdk1 substrate is myosin phosphatase targeting subunit 1 (MYPT1), and we show that MYPT1 localization to kinetochores depends on Cyclin A/Cdk1 activity and that MYPT1 destabilizes k-MT attachments by negatively regulating Plk1 at kinetochores. Thus, Cyclin A/Cdk1 phosphorylation primes MYPT1 for Plk1 binding. Interestingly, priming of PBIP1 by Plk1 itself (self-priming) increased in MYPT1-depleted cells showing that MYPT1 provides a molecular link between the processes of Cdk1-dependent priming and self-priming of Plk1 substrates. These data demonstrate cross-regulation between Cyclin A/Cdk1-dependent and Plk1-dependent phosphorylation of substrates during mitosis to ensure efficient correction of k-MT attachment errors necessary for high mitotic fidelity.

    更新日期:2017-11-20
  • Cocaine-induced adaptation of dopamine D2S, but not D2L autoreceptors
    eLife (IF 7.725) Pub Date : 2017-11-20
    Brooks G Robinson, Alec F Condon, Daniela Radl, Emiliana Borrelli, John T Williams, Kim A Neve

    The dopamine D2 receptor has two splice variants, D2S (Short) and D2L (Long). In dopamine neurons, both variants can act as autoreceptors to regulate neuronal excitability and dopamine release, but the roles of each variant are incompletely characterized. In a previous study we used viral receptor expression in D2 receptor knockout mice to show distinct effects of calcium signaling on D2S and D2L autoreceptor function (Gantz et al., 2015). However, the cocaine-induced plasticity of D2 receptor desensitization observed in wild type mice was not recapitulated with this method of receptor expression. Here we use mice with genetic knockouts of either the D2S or D2L variant to investigate cocaine-induced plasticity in D2 receptor signaling. Following a single in vivo cocaine exposure, the desensitization of D2 receptors from neurons expressing only the D2S variant was reduced. This did not occur in D2L-expressing neurons, indicating differential drug-induced plasticity between the variants. https://doi.org/10.7554/eLife.31924.001

    更新日期:2017-11-20
  • Ribosome surface properties may impose limits on the nature of the cytoplasmic proteome
    eLife (IF 7.725) Pub Date : 2017-11-20
    Paul E Schavemaker, Wojciech M Śmigiel, Bert Poolman

    Much of the molecular motion in the cytoplasm is diffusive, which possibly limits the tempo of processes. We studied the dependence of protein mobility on protein surface properties and ionic strength. We used surface-modified fluorescent proteins (FPs) and determined their translational diffusion coefficients (D) in the cytoplasm of Escherichia coli, Lactococcus lactis and Haloferax volcanii. We find that in E. coli D depends on the net charge and its distribution over the protein, with positive proteins diffusing up to 100-fold slower than negative ones. This effect is weaker in L. lactis and Hfx. volcanii due to electrostatic screening. The decrease in mobility is probably caused by interaction of positive FPs with ribosomes as shown in in vivo diffusion measurements and confirmed in vitro with purified ribosomes. Ribosome surface properties may thus limit the composition of the cytoplasmic proteome. This finding lays bare a paradox in the functioning of prokaryotic (endo)symbionts.

    更新日期:2017-11-20
  • Horizontal Gene Transfer: Learning from losers
    eLife (IF 7.725) Pub Date : 2017-11-17
    Benjamin Kirkup

    Bacteria can overcome environmental challenges by killing nearby bacteria and incorporating their DNA.

    更新日期:2017-11-19
  • The structure of the COPI coat determined within the cell
    eLife (IF 7.725) Pub Date : 2017-11-17
    Yury S Bykov, Miroslava Schaffer, Svetlana O Dodonova, Sahradha Albert, Jürgen M Plitzko, Wolfgang Baumeister, Benjamin D Engel, John AG Briggs

    COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. In a previous paper (Dodonova et al., 2017), we determined a complete structural model of the in vitro reconstituted COPI coat. Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β'-COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

    更新日期:2017-11-17
  • Autophagic cell death is dependent on lysosomal membrane permeability through Bax and Bak
    eLife (IF 7.725) Pub Date : 2017-11-17
    Jason Karch, Tobias G Schips, Bryan D Maliken, Matthew J Brody, Michelle A Sargent, Onur Kanisciak, Jeffery D Molkentin

    Cells deficient in the pro-death Bcl-2 family members Bax and Bak are known to be resistant to apoptotic cell death, and in a previous eLIFE paper, Karch et al., 2013 showed that these 2 effectors are also needed for mitochondrial-dependent cellular necrosis. Here we show that mouse embryonic fibroblasts deficient in Bax/Bak1 are resistant to the third major form of cell death associated with autophagy through a mechanism involving lysosome permeability. Indeed, specifically targeting Bax only to the lysosome restores autophagic cell death in Bax/Bak1 null cells. Moreover, a monomeric-only mutant form of Bax is sufficient to increase lysosomal membrane permeability and restore autophagic cell death in Bax/Bak1 double-deleted mouse embryonic fibroblasts. Finally, increasing lysosomal permeability through a lysomotropic detergent in cells devoid of Bax/Bak1 restores autophagic cell death, collectively indicting that Bax/Bak integrate all major forms of cell death through direct effects on membrane permeability of multiple intracellular organelles.

    更新日期:2017-11-17
  • Stabilization and structural analysis of a membrane-associated hIAPP aggregation intermediate
    eLife (IF 7.725) Pub Date : 2017-11-17
    Diana C Rodriguez Camargo, Kyle J Korshavn, Alexander Jussupow, Kolio Raltchev, David Goricanec, Markus Fleisch, Riddhiman Sarkar, Kai Xue, Michaela Aichler, Gabriele Mettenleiter, Axel Karl Walch, Carlo Camilloni, Franz Hagn, Bernd Reif, Ayyalusamy Ramamoorthy

    Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique b-strand structure distinct from the conventional amyloid b-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.

    更新日期:2017-11-17
  • Revealing the distribution of transmembrane currents along the dendritic tree of a neuron from extracellular recordings
    eLife (IF 7.725) Pub Date : 2017-11-17
    Dorottya Cserpán, Domokos Meszéna, Lucia Wittner, Kinga Tóth, István Ulbert, Zoltán Somogyvári, Daniel K Wojcik

    Revealing the current source distribution along the neuronal membrane is a key step on the way to understanding neural computations, however, the experimental and theoretical tools to achieve sufficient spatiotemporal resolution for the estimation remain to be established. Here we address this problem using extracellularly recorded potentials with arbitrarily distributed electrodes for a neuron of known morphology. We use simulations of models with varying complexity to validate the proposed method and to give recommendations for experimental applications. The method is applied to in vitro data from rat hippocampus.

    更新日期:2017-11-17
  • The somatically generated portion of the T cell receptor CDR3α contributes to the MHC allele specificity of the T cell receptor
    eLife (IF 7.725) Pub Date : 2017-11-17
    Philippa Marrack, Sai Harsha Krovi, Daniel Silberman, Janice White, Eleanor Kushnir, Maki Nakayama, James Crooks, Thomas Danhorn, Sonia Leach, Randy Anselment, James Scott-Browne, Laurent Gapin, John Kappler

    Mature T cells bearing αβ T cell receptors react with foreign antigens bound to alleles of major histocompatibility complex proteins (MHC) that they were exposed to during their development in the thymus, a phenomenon known as positive selection. The structural basis for positive selection has long been debated. Here, using mice expressing one of two different T cell receptor β chains and various MHC alleles, we show that positive selection-induced MHC bias of T cell receptors is affected both by the germline encoded elements of the T cell receptor α and β chain and, surprisingly, dramatically affected by the non germ line encoded portions of CDR3 of the T cell receptor α chain. Thus, in addition to determining specificity for antigen, the non germline encoded elements of T cell receptors may help the proteins cope with the extremely polymorphic nature of major histocompatibility complex products within the species.

    更新日期:2017-11-17
  • Convergence of topological domain boundaries, insulators, and polytene interbands revealed by high-resolution mapping of chromatin contacts in the early Drosophila melanogaster embryo
    eLife (IF 7.725) Pub Date : 2017-11-17
    Michael R Stadler, Jenna E Haines, Michael Eisen

    High-throughput assays of three-dimensional interactions of chromosomes have shed considerable light on the structure of animal chromatin. Despite this progress, the precise physical nature of observed structures and the forces that govern their establishment remain poorly understood. Here we present high resolution Hi-C data from early Drosophila embryos. We demonstrate that boundaries between topological domains of various sizes map to DNA elements that resemble classical insulator elements: short genomic regions sensitive to DNase digestion that are strongly bound by known insulator proteins and are frequently located between divergent promoters. Further, we show a striking correspondence between these elements and the locations of mapped polytene interband regions. We believe it is likely this relationship between insulators, topological boundaries, and polytene interbands extends across the genome, and we therefore propose a model in which decompaction of boundary-insulator-interband regions drives the organization of interphase chromosomes by creating stable physical separation between adjacent domains.

    更新日期:2017-11-17
  • Rapid DNA replication origin licensing protects stem cell pluripotency
    eLife (IF 7.725) Pub Date : 2017-11-17
    Jacob Peter Matson, Raluca Dumitru, Philip Coryell, Ryan M Baxley, Weili Chen, Kirk Twaroski, Beau R Webber, Jakub Tolar, Anja-Katrin Bielinsky, Jeremy E Purvis, Jeanette Gowen Cook

    Complete and robust human genome duplication requires loading MCM helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single cell analyses we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation towards all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

    更新日期:2017-11-17
  • Revealing the distribution of transmembrane currents along the dendritic tree of a neuron from extracellular recordings
    eLife (IF 7.725) Pub Date : 2017-11-17
    Dorottya Cserpán, Domokos Meszéna, Lucia Wittner, Kinga Tóth, István Ulbert, Zoltán Somogyvári, Daniel K Wojcik

    Revealing the current source distribution along the neuronal membrane is a key step on the way to understanding neural computations, however, the experimental and theoretical tools to achieve sufficient spatiotemporal resolution for the estimation remain to be established. Here we address this problem using extracellularly recorded potentials with arbitrarily distributed electrodes for a neuron of known morphology. We use simulations of models with varying complexity to validate the proposed method and to give recommendations for experimental applications. The method is applied to in vitro data from rat hippocampus.

    更新日期:2017-11-17
  • Stabilization and structural analysis of a membrane-associated hIAPP aggregation intermediate
    eLife (IF 7.725) Pub Date : 2017-11-17
    Diana C Rodriguez Camargo, Kyle J Korshavn, Alexander Jussupow, Kolio Raltchev, David Goricanec, Markus Fleisch, Riddhiman Sarkar, Kai Xue, Michaela Aichler, Gabriele Mettenleiter, Axel Karl Walch, Carlo Camilloni, Franz Hagn, Bernd Reif, Ayyalusamy Ramamoorthy

    Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique b-strand structure distinct from the conventional amyloid b-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.

    更新日期:2017-11-17
  • The structure of the COPI coat determined within the cell
    eLife (IF 7.725) Pub Date : 2017-11-17
    Yury S Bykov, Miroslava Schaffer, Svetlana O Dodonova, Sahradha Albert, Jürgen M Plitzko, Wolfgang Baumeister, Benjamin D Engel, John AG Briggs

    COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. In a previous paper (Dodonova et al., 2017), we determined a complete structural model of the in vitro reconstituted COPI coat. Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β'-COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

    更新日期:2017-11-17
  • The somatically generated portion of the T cell receptor CDR3α contributes to the MHC allele specificity of the T cell receptor
    eLife (IF 7.725) Pub Date : 2017-11-17
    Philippa Marrack, Sai Harsha Krovi, Daniel Silberman, Janice White, Eleanor Kushnir, Maki Nakayama, James Crooks, Thomas Danhorn, Sonia Leach, Randy Anselment, James Scott-Browne, Laurent Gapin, John Kappler

    Mature T cells bearing αβ T cell receptors react with foreign antigens bound to alleles of major histocompatibility complex proteins (MHC) that they were exposed to during their development in the thymus, a phenomenon known as positive selection. The structural basis for positive selection has long been debated. Here, using mice expressing one of two different T cell receptor β chains and various MHC alleles, we show that positive selection-induced MHC bias of T cell receptors is affected both by the germline encoded elements of the T cell receptor α and β chain and, surprisingly, dramatically affected by the non germ line encoded portions of CDR3 of the T cell receptor α chain. Thus, in addition to determining specificity for antigen, the non germline encoded elements of T cell receptors may help the proteins cope with the extremely polymorphic nature of major histocompatibility complex products within the species.

    更新日期:2017-11-17
  • Convergence of topological domain boundaries, insulators, and polytene interbands revealed by high-resolution mapping of chromatin contacts in the early Drosophila melanogaster embryo
    eLife (IF 7.725) Pub Date : 2017-11-17
    Michael R Stadler, Jenna E Haines, Michael Eisen

    High-throughput assays of three-dimensional interactions of chromosomes have shed considerable light on the structure of animal chromatin. Despite this progress, the precise physical nature of observed structures and the forces that govern their establishment remain poorly understood. Here we present high resolution Hi-C data from early Drosophila embryos. We demonstrate that boundaries between topological domains of various sizes map to DNA elements that resemble classical insulator elements: short genomic regions sensitive to DNase digestion that are strongly bound by known insulator proteins and are frequently located between divergent promoters. Further, we show a striking correspondence between these elements and the locations of mapped polytene interband regions. We believe it is likely this relationship between insulators, topological boundaries, and polytene interbands extends across the genome, and we therefore propose a model in which decompaction of boundary-insulator-interband regions drives the organization of interphase chromosomes by creating stable physical separation between adjacent domains.

    更新日期:2017-11-17
  • Small molecule Photoregulin3 prevents retinal degeneration in the RhoP23H mouse model of retinitis pigmentosa
    eLife (IF 7.725) Pub Date : 2017-11-17
    Paul A Nakamura, Andy A Shimchuk, Shibing Tang, Zhizhi Wang, Kole DeGolier, Sheng Ding, Thomas A Reh

    Regulation of rod gene expression has emerged as a potential therapeutic strategy to treat retinal degenerative diseases like retinitis pigmentosa (RP). We previously reported on a small molecule modulator of the rod transcription factor Nr2e3, Photoregulin1 (PR1), that regulates the expression of photoreceptor-specific genes. Although PR1 slows the progression of retinal degeneration in models of RP in vitro, in vivo analyses were not possible with PR1. We now report a structurally unrelated compound, Photoregulin3 (PR3) that also inhibits rod photoreceptor gene expression, potentially though Nr2e3 modulation. To determine the effectiveness of PR3 as a potential therapy for RP, we treated RhoP23H mice with PR3 and assessed retinal structure and function. PR3-treated RhoP23H mice showed significant structural and functional photoreceptor rescue compared with vehicle-treated littermate control mice. These results provide further support that pharmacological modulation of rod gene expression provides a potential strategy for the treatment of RP. https://doi.org/10.7554/eLife.30577.001

    更新日期:2017-11-17
  • Rapid DNA replication origin licensing protects stem cell pluripotency
    eLife (IF 7.725) Pub Date : 2017-11-17
    Jacob Peter Matson, Raluca Dumitru, Philip Coryell, Ryan M Baxley, Weili Chen, Kirk Twaroski, Beau R Webber, Jakub Tolar, Anja-Katrin Bielinsky, Jeremy E Purvis, Jeanette Gowen Cook

    Complete and robust human genome duplication requires loading MCM helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single cell analyses we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation towards all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

    更新日期:2017-11-17
  • Autophagic cell death is dependent on lysosomal membrane permeability through Bax and Bak
    eLife (IF 7.725) Pub Date : 2017-11-17
    Jason Karch, Tobias G Schips, Bryan D Maliken, Matthew J Brody, Michelle A Sargent, Onur Kanisciak, Jeffery D Molkentin

    Cells deficient in the pro-death Bcl-2 family members Bax and Bak are known to be resistant to apoptotic cell death, and in a previous eLIFE paper, Karch et al., 2013 showed that these 2 effectors are also needed for mitochondrial-dependent cellular necrosis. Here we show that mouse embryonic fibroblasts deficient in Bax/Bak1 are resistant to the third major form of cell death associated with autophagy through a mechanism involving lysosome permeability. Indeed, specifically targeting Bax only to the lysosome restores autophagic cell death in Bax/Bak1 null cells. Moreover, a monomeric-only mutant form of Bax is sufficient to increase lysosomal membrane permeability and restore autophagic cell death in Bax/Bak1 double-deleted mouse embryonic fibroblasts. Finally, increasing lysosomal permeability through a lysomotropic detergent in cells devoid of Bax/Bak1 restores autophagic cell death, collectively indicting that Bax/Bak integrate all major forms of cell death through direct effects on membrane permeability of multiple intracellular organelles.

    更新日期:2017-11-17
  • Atg9 antagonizes TOR signaling to regulate intestinal cell growth and epithelial homeostasis in Drosophila
    eLife (IF 7.725) Pub Date : 2017-11-16
    Jung-Kun Wen, Yi-Ting Wang, Chih-Chiang Chan, Cheng-Wen Hsieh, Hsiao-Man Liao, Chin-Chun Hung, Guang-Chao Chen

    Autophagy is essential for maintaining cellular homeostasis and survival under various stress conditions. Autophagy-related gene 9 (Atg9) encodes a multipass transmembrane protein thought to act as a membrane carrier for forming autophagosomes. However, the molecular regulation and physiological importance of Atg9 in animal development remain largely unclear. Here, we generated Atg9 null mutant flies and found that loss of Atg9 led to shortened lifespan, locomotor defects, and increased susceptibility to stress. Atg9 loss also resulted in aberrant adult midgut morphology with dramatically enlarged enterocytes. Interestingly, inhibiting the TOR signaling pathway rescued the midgut defects of the Atg9 mutants. In addition, Atg9 interacted with PALS1-associated tight junction protein (Patj), which associates with TSC2 to regulate TOR activity. Depletion of Atg9 caused a marked decrease in TSC2 levels. Our findings revealed an antagonistic relationship between Atg9 and TOR signaling in the regulation of cell growth and tissue homeostasis. https://doi.org/10.7554/eLife.29338.001

    更新日期:2017-11-17
  • LRP1 integrates murine macrophage cholesterol homeostasis and inflammatory responses in atherosclerosis
    eLife (IF 7.725) Pub Date : 2017-11-16
    Xunde Xian, Yinyuan Ding, Marco Dieckmann, Li Zhou, Florian Plattner, Mingxia Liu, John S Parks, Robert E Hammer, Philippe Boucher, Shirling Tsai, Joachim Herz

    Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional cell surface receptor with diverse physiological roles, ranging from cellular uptake of lipoproteins and other cargo by endocytosis to sensor of the extracellular environment and integrator of a wide range of signaling mechanisms. As a chylomicron remnant receptor, LRP1 controls systemic lipid metabolism in concert with the LDL receptor in the liver, whereas in smooth muscle cells (SMC) LRP1 functions as a co-receptor for TGFβ and PDGFRβ in reverse cholesterol transport and the maintenance of vascular wall integrity. Here we used a knockin mouse model to uncover a novel atheroprotective role for LRP1 in macrophages where tyrosine phosphorylation of an NPxY motif in its intracellular domain initiates a signaling cascade along an LRP1/SHC1/PI3K/AKT/PPARγ/LXR axis to regulate and integrate cellular cholesterol homeostasis through the expression of the major cholesterol exporter ABCA1 with apoptotic cell removal and inflammatory responses. https://doi.org/10.7554/eLife.29292.001

    更新日期:2017-11-17
  • A high-resolution mRNA expression time course of embryonic development in zebrafish
    eLife (IF 7.725) Pub Date : 2017-11-16
    Richard J White, John E Collins, Ian M Sealy, Neha Wali, Christopher M Dooley, Zsofia Digby, Derek L Stemple, Daniel N Murphy, Konstantinos Billis, Thibaut Hourlier, Anja Füllgrabe, Matthew P Davis, Anton J Enright, Elisabeth M Busch-Nentwich

    We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3′ end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3′ ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl. https://doi.org/10.7554/eLife.30860.001

    更新日期:2017-11-17
  • Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis
    eLife (IF 7.725) Pub Date : 2017-11-10
    Martin Steger, Federico Diez, Herschel S Dhekne, Pawel Lis, Raja S Nirujogi, Ozge Karayel, Francesca Tonelli, Terina N Martinez, Esben Lorentzen, Suzanne R Pfeffer, Dario R Alessi, Matthias Mann

    We previously reported that Parkinson's disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger, Tonelli et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

    更新日期:2017-11-16
  • Pharmacological augmentation of nicotinamide phosphoribosyltransferase (NAMPT) protects against paclitaxel-induced peripheral neuropathy
    eLife (IF 7.725) Pub Date : 2017-11-10
    Peter M LoCoco, April L Risinger, Hudson R Smith, Teresa S Chavera, Kelly A Berg, William P Clarke

    Chemotherapy-induced peripheral neuropathy (CIPN) arises from collateral damage to peripheral afferent sensory neurons by anticancer pharmacotherapy, leading to debilitating neuropathic pain. No effective treatment for CIPN exists, short of dose-reduction which worsens cancer prognosis. Here we report that stimulation of nicotinamide phosphoribosyltransferase (NAMPT) produced robust neuroprotection in an aggressive CIPN model utilizing the frontline anticancer drug, paclitaxel (PTX). Daily treatment of rats with the first-in-class NAMPT stimulator, P7C3-A20, prevented behavioral and histologic indicators of peripheral neuropathy, stimulated tissue NAD recovery, improved general health, and abolished attrition produced by a near maximum-tolerated dose of PTX. Inhibition of NAMPT blocked P7C3-A20-mediated neuroprotection, whereas supplementation with the NAMPT substrate, nicotinamide, potentiated a subthreshold dose of P7C3-A20 to full efficacy. Importantly, P7C3-A20 blocked PTX-induced allodynia in tumored mice without reducing antitumoral efficacy. These findings identify enhancement of NAMPT activity as a promising new therapeutic strategy to protect against anticancer drug-induced peripheral neurotoxicity.

    更新日期:2017-11-16
  • Structural basis of host recognition and biofilm formation by Salmonella Saf pili
    eLife (IF 7.725) Pub Date : 2017-11-10
    Longhui Zeng, Li Zhang, Pengran Wang, Guoyu Meng

    Pili are critical in host recognition, colonization and biofilm formation during bacterial infection. Here, we report the crystal structures of SafD-dsc and SafD-SafA-SafA (SafDAA-dsc) in Saf pili. Cell adherence assays show that SafD and SafA are both required for host recognition, suggesting a poly-adhesive mechanism for Saf pili. Moreover, the SafDAA-dsc structure, as well as SAXS characterization, reveals an unexpected inter-molecular oligomerization, prompting the investigation of Saf-driven self-association in biofilm formation. The bead/cell aggregation and biofilm formation assays are used to demonstrate the novel function of Saf pili. Structure-based mutants targeting the inter-molecular hydrogen bonds and complementary architecture/surfaces in SafDAA-dsc dimers significantly impaired the Saf self-association activity and biofilm formation. In summary, our results identify two novel functions of Saf pili: the poly-adhesive and self-associating activities. More importantly, Saf-Saf structures and functional characterizations help to define a pili-mediated inter-cellular oligomerizaiton mechanism for bacterial aggregation, colonization and ultimate biofilm formation.

    更新日期:2017-11-16
  • Ubiquitin turnover and endocytic trafficking in yeast are regulated by Ser57 phosphorylation of ubiquitin
    eLife (IF 7.725) Pub Date : 2017-11-13
    Sora Lee, Jessica M Tumolo, Aaron C Ehlinger, Kristin K Jernigan, Susan J Qualls-Histed, Pi-Chiang Hsu, W Hayes McDonald, Walter J Chazin, Jason A MacGurn

    Despite its central role in protein degradation little is known about the molecular mechanisms that sense, maintain, and regulate steady state concentration of ubiquitin in the cell. Here, we describe a novel mechanism for regulation of ubiquitin homeostasis that is mediated by phosphorylation of ubiquitin at the Ser57 position. We find that loss of Ppz phosphatase activity leads to defects in ubiquitin homeostasis that are at least partially attributable to elevated levels of Ser57 phosphorylated ubiquitin. Phosphomimetic mutation at the Ser57 position of ubiquitin conferred increased rates of endocytic trafficking and ubiquitin turnover. These phenotypes are associated with bypass of recognition by endosome-localized deubiquitylases - including Doa4 which is critical for regulation of ubiquitin recycling. Thus, ubiquitin homeostasis is significantly impacted by the rate of ubiquitin flux through the endocytic pathway and by signaling pathways that converge on ubiquitin itself to determine whether it is recycled or degraded in the vacuole.

    更新日期:2017-11-16
  • Simultaneous enumeration of cancer and immune cell types from bulk tumor gene expression data
    eLife (IF 7.725) Pub Date : 2017-11-13
    Julien Racle, Kaat de Jonge, Petra Baumgaertner, Daniel E Speiser, David Gfeller

    Immune cells infiltrating tumors can have important impact on tumor progression and response to therapy. We present an efficient algorithm to simultaneously estimate the fraction of cancer and immune cell types from bulk tumor gene expression data. Our method integrates novel gene expression profiles from each major non-malignant cell type found in tumors, renormalization based on cell-type specific mRNA content, and the ability to consider uncharacterized and possibly highly variable cell types. Feasibility is demonstrated by validation with flow cytometry, immunohistochemistry and single-cell RNA-Seq analyses of human melanoma and colorectal tumor specimens. Altogether, our work not only improves accuracy but also broadens the scope of absolute cell fraction predictions from tumor gene expression data, and provides a unique novel experimental benchmark for immunogenomics analyses in cancer research.

    更新日期:2017-11-16
  • Regulatory network structure determines patterns of intermolecular epistasis
    eLife (IF 7.725) Pub Date : 2017-11-13
    Mato Lagator, Srdjan Sarikas, Hande Acar, Jonathan P Bollback, Călin C Guet

    Most phenotypes are determined by molecular systems composed of specifically interacting molecules. However, unlike for individual components, little is known about the distributions of mutational effects of molecular systems as a whole. We ask how the distribution of mutational effects of a transcriptional regulatory system differs from the distributions of its components, by first independently, and then simultaneously, mutating a transcription factor and the associated promoter it represses. We find that the system distribution exhibits increased phenotypic variation compared to individual component distributions - an effect arising from intermolecular epistasis between the transcription factor and its DNA-binding site. In large part, this epistasis can be qualitatively attributed to the structure of the transcriptional regulatory system, and could therefore be a common feature in prokaryotes. Counter-intuitively, intermolecular epistasis can alleviate the constraints of individual components, thereby increasing phenotypic variation that selection could act on and facilitating adaptive evolution.

    更新日期:2017-11-16
  • Defective synaptic transmission causes disease signs in a mouse model of Juvenile Neuronal Ceroid Lipofuscinosis
    eLife (IF 7.725) Pub Date : 2017-11-14
    Benedikt Grünewald, Maren D Lange, Christian Werner, Aet O'Leary, Andreas Weishaupt, Sandy Popp, David A Pearce, Heinz Wiendl, Andreas Reif, Hans C Pape, Klaus V Toyka, Claudia Sommer, Christian Geis

    Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease) caused by mutations in the CLN3 gene is the most prevalent inherited neurodegenerative disease in childhood resulting in widespread central nervous system dysfunction and premature death. The consequences of CLN3 mutation on the progression of the disease, on neuronal transmission, and on central nervous network dysfunction are poorly understood. We used Cln3 knockout (Cln3Δex7/8) mice and found increased anxiety-related behavior and impaired aversive learning as well as markedly affected motor function including disordered coordination. Patch-clamp and loose-patch recordings revealed severely affected inhibitory and excitatory synaptic transmission in amygdala, hippocampus, and in cerebellar networks. Changes in presynaptic release properties may result from dysfunction of CLN3 protein. Furthermore, loss of calbindin, neuropeptide Y, parvalbumin, and GAD65-positive interneurons in central networks collectively support the hypothesis that degeneration of GABAergic interneurons may be the cause of supraspinal GABAergic disinhibition.

    更新日期:2017-11-16
  • Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals
    eLife (IF 7.725) Pub Date : 2017-11-14
    Inge Kühl, Maria Miranda, Ilian Atanassov, Irina Kuznetsova, Yvonne Hinze, Arnaud Mourier, Aleksandra Filipovska, Nils-Göran Larsson

    Dysfunction of the oxidative phosphorylation (OXPHOS) system is a major cause of human disease and the cellular consequences are highly complex. Here, we present comparative analyses of mitochondrial proteomes, cellular transcriptomes and targeted metabolomics of five knockout mouse strains deficient in essential factors required for mitochondrial DNA gene expression, leading to OXPHOS dysfunction. Moreover, we describe sequential protein changes during post-natal development and progressive OXPHOS dysfunction in time course analyses in control mice and a middle lifespan knockout, respectively. Very unexpectedly, we identify a new response pathway to OXPHOS dysfunction in which the intra-mitochondrial synthesis of coenzyme Q (ubiquinone, Q) and Q levels are profoundly decreased, pointing towards novel possibilities for therapy. Our extensive omics analyses provide a high-quality resource of altered gene expression patterns under severe OXPHOS deficiency comparing several mouse models, that will deepen our understanding, open avenues for research and provide an important reference for diagnosis and treatment.

    更新日期:2017-11-16
  • An adaptation of astronomical image processing enables characterization and functional 3D mapping of individual sites of excitation-contraction coupling in rat cardiac muscle
    eLife (IF 7.725) Pub Date : 2017-11-14
    Qinghai Tian, Lars Kaestner, Laura Schröder, Jia Guo, Peter Lipp

    In beating cardiomyocytes, synchronized localized Ca2+ transients from thousands of active excitation-contraction coupling sites (ECC couplons) comprising plasma and sarcoplasmic reticulum membrane calcium channels are important determinants of the heart's performance. Nevertheless, our knowledge about their properties is limited by the lack of appropriate experimental and analysis strategies. We designed CaCLEAN to untangle fundamental characteristics of ECC couplons by combining the astronomer's CLEAN algorithm with known properties of calcium diffusion. CaCLEAN empowers the investigation of fundamental properties of ECC couplons in beating cardiomyocytes without pharmacological interventions. On the nanoscopic level of individual ECC couplons, we reveal their role in the negative amplitude-frequency relationship and b-adrenergic stimulation, including decreasing and increasing firing reliability, respectively. CaCLEAN combined with 3D confocal imaging of beating cardiomyocytes provides a functional 3D map of active ECC couplons (on average 17.000 per myocyte). CaCLEAN will further enlighten remodelling processes of ECC couplons underlying cardiac diseases.

    更新日期:2017-11-16
  • Glutamate is required for depression but not potentiation of long-term presynaptic function
    eLife (IF 7.725) Pub Date : 2017-11-15
    Zahid Padamsey, Rudi Tong, Nigel Emptage

    Hebbian plasticity is thought to require glutamate signalling. We show this is not the case for hippocampal presynaptic long-term potentiation (LTPpre), which is expressed as an increase in transmitter release probability (Pr). We find that LTPpre can be induced by pairing pre- and postsynaptic spiking in the absence of glutamate signalling. LTPpre induction involves a non-canonical mechanism of retrograde nitric oxide signalling, which is triggered by Ca2+ influx from L-type voltage-gated Ca2+ channels, not postsynaptic NMDA receptors (NMDARs), and does not require glutamate release. When glutamate release occurs, it decreases Pr by activating presynaptic NMDARs, and promotes presynaptic long-term depression. Net changes in Pr, therefore, depend on two opposing factors: 1) Hebbian activity, which increases Pr, and 2) glutamate release, which decreases Pr. Accordingly, release failures during Hebbian activity promote LTPpre induction. Our findings reveal a novel framework of presynaptic plasticity that radically differs from traditional models of postsynaptic plasticity.

    更新日期:2017-11-16
  • Bacterial fumarase and L-malic acid are evolutionary ancient components of the DNA damage response
    eLife (IF 7.725) Pub Date : 2017-11-15
    Esti Singer, Yardena BH Silas, Sigal Ben-Yehuda, Ophry Pines

    Fumarase is distributed between two compartments of the eukaryotic cell. The enzyme catalyses the reversible conversion of fumaric to L-malic acid in mitochondria as part of the tricarboxylic acid (TCA) cycle, and in the cytosol/nucleus as part of the DNA damage response (DDR). Here we show that fumarase of the model prokaryote Bacillus subtilis (Fum-bc) is induced upon DNA damage, co-localized with the bacterial DNA and is required for the DDR. Fum-bc can substitute for both eukaryotic functions in yeast. Furthermore, we found that the fumarase dependent intracellular signaling of the B. subtilis DDR is achieved via production of L-malic acid, which affects the translation of RecN, the first protein recruited to DNA damage sites. This study provides a different evolutionary scenario in which the dual function of the ancient prokaryotic fumarase, led to its subsequent distribution into different cellular compartments in eukaryotes.

    更新日期:2017-11-16
  • An Eya1-Notch axis specifies bipotential epibranchial differentiation in mammalian craniofacial morphogenesis
    eLife (IF 7.725) Pub Date : 2017-11-15
    Haoran Zhang, Li Wang, Elaine Yee Man Wong, Sze Lan Tsang, Pin-Xian Xu, Urban Lendahl, Mai Har Sham

    Craniofacial morphogenesis requires proper development of pharyngeal arches and epibranchial placodes. We show that the epibranchial placodes, in addition to giving rise to cranial sensory neurons, generate a novel lineage-related non-neuronal cell population for mouse pharyngeal arch development. Eya1 is essential for the development of epibranchial placodes and proximal pharyngeal arches. We identify an Eya1-Notch regulatory axis that specifies both the neuronal and non-neuronal commitment of the epibranchial placode, where Notch acts downstream of Eya1 and promotes the non-neuronal cell fate. Notch is regulated by the threonine phosphatase activity of Eya1. Eya1 dephosphorylates p-threonine-2122 of the Notch1 intracellular domain (Notch1 ICD), which increases the stability of Notch1 ICD and maintains Notch signaling activity in the non-neuronal epibranchial placodal cells. Our data unveil a more complex differentiation program in epibranchial placodes and an important role for the Eya1-Notch axis in craniofacial morphogenesis.

    更新日期:2017-11-16
  • Subcellular analysis of pigeon hair cells implicates vesicular trafficking in cuticulosome formation and maintenance
    eLife (IF 7.725) Pub Date : 2017-11-15
    Simon Nimpf, Erich Pascal Malkemper, Mattias Lauwers, Lyubov Ushakova, Gregory Nordmann, Andrea Wenninger-Weinzierl, Thomas R Burkard, Sonja Jacob, Thomas Heuser, Guenter P Resch, David A Keays

    Hair cells are specialized sensors located in the inner ear that enable the transduction of sound, motion, and gravity into neuronal impulses. In birds some hair cells contain an iron-rich organelle, the cuticulosome, that has been implicated in the magnetic sense. Here, we exploit histological, transcriptomic and tomographic methods to investigate the development of cuticulosomes, as well as the molecular and subcellular architecture of cuticulosome positive hair cells. We show that this organelle forms rapidly after hatching in a process that involves vesicle fusion and nucleation of ferritin nanoparticles. We further report that transcripts involved in endocytosis, extracellular exosomes, and metal ion binding are differentially expressed in cuticulosome positive hair cells. These data suggest that the cuticulosome and the associated molecular machinery regulate the concentration of iron within the labyrinth of the inner ear, which might indirectly tune a magnetic sensor that relies on electromagnetic induction.

    更新日期:2017-11-16
  • Vg1-Nodal heterodimers are the endogenous inducers of mesendoderm
    eLife (IF 7.725) Pub Date : 2017-11-15
    Tessa Grace Montague, Alexander F Schier

    Nodal is considered the key inducer of mesendoderm in vertebrate embryos and embryonic stem cells. Other TGF-beta-related signals, such as Vg1/Dvr1/Gdf3, have also been implicated in this process but their roles have been unclear or controversial. Here we report that zebrafish embryos without maternally provided vg1 fail to form endoderm and head and trunk mesoderm, and closely resemble nodal loss-of-function mutants. Although Nodal is processed and secreted without Vg1, it requires Vg1 for its endogenous activity. Conversely, Vg1 is unprocessed and resides in the endoplasmic reticulum without Nodal, and is only secreted, processed and active in the presence of Nodal. Co-expression of Nodal and Vg1 results in heterodimer formation and mesendoderm induction. Thus, mesendoderm induction relies on the combination of two TGF-beta-related signals: maternal and ubiquitous Vg1, and zygotic and localized Nodal. Modeling reveals that the pool of maternal Vg1 enables rapid signaling at low concentrations of zygotic Nodal.

    更新日期:2017-11-16
  • DDM1 and Lsh remodelers allow methylation of DNA wrapped in nucleosomes
    eLife (IF 7.725) Pub Date : 2017-11-15
    David B Lyons, Daniel Zilberman

    Cytosine methylation regulates essential genome functions across eukaryotes, but the fundamental question of whether nucleosomal or naked DNA is the preferred substrate of plant and animal methyltransferases remains unresolved. Here, we show that genetic inactivation of a single DDM1/Lsh family nucleosome remodeler biases methylation toward inter-nucleosomal linker DNA in Arabidopsis thaliana and mouse. We find that DDM1 enables methylation of DNA bound to the nucleosome, suggesting that nucleosome-free DNA is the preferred substrate of eukaryotic methyltransferases in vivo. Furthermore, we show that simultaneous mutation of DDM1 and linker histone H1 in Arabidopsis reproduces the strong linker-specific methylation patterns of species that diverged from flowering plants and animals over a billion years ago. Our results indicate that in the absence of remodeling, nucleosomes are strong barriers to DNA methyltransferases. Linker-specific methylation can evolve simply by breaking the connection between nucleosome remodeling and DNA methylation.

    更新日期:2017-11-16
  • Gdf3 is required for robust Nodal signaling during germ layer formation and left-right patterning
    eLife (IF 7.725) Pub Date : 2017-11-15
    Jose L Pelliccia, Granton A Jindal, Rebecca D Burdine

    Vertebrate embryonic patterning depends on signaling from Nodal, a TGFβ superfamily member. There are three Nodal orthologs in zebrafish; southpaw directs left-right asymmetries, while squint and cyclops function earlier to pattern mesendoderm. TGFβ member Vg1 is implicated in mesoderm formation but the role of the zebrafish ortholog, Growth differentiation factor 3 (Gdf3), has not been fully explored. We show that zygotic expression of gdf3 is dispensable for embryonic development, while maternally deposited gdf3 is required for mesendoderm formation and dorsal-ventral patterning. We further show that Gdf3 can affect left-right patterning at multiple stages, including proper development of regional cell morphology in Kupffer's vesicle and the establishment of southpaw expression in the lateral plate mesoderm. Collectively, our data indicate that gdf3 is critical for robust Nodal signaling at multiple stages in zebrafish embryonic development.

    更新日期:2017-11-16
  • A versatile genetic tool for post-translational control of gene expression in Drosophila melanogaster
    eLife (IF 7.725) Pub Date : 2017-11-15
    Sachin Sethi, Jing W Wang

    Several techniques have been developed to manipulate gene expression temporally in intact neural circuits. However, the applicability of current tools developed for in vivo studies in Drosophila is limited by their incompatibility with existing GAL4 lines and side effects on physiology and behavior. To circumvent these limitations, we adopted a strategy to reversibly regulate protein degradation with a small molecule by using a destabilizing domain (DD). We show that this system is effective across different tissues and developmental stages. We further show that this system can be used to control in vivo gene expression levels with low background, large dynamic range, and in a reversible manner without detectable side effects on the lifespan or behavior of the animal. Additionally, we engineered tools for chemically controlling gene expression (GAL80-DD) and recombination (FLP-DD). We demonstrate the applicability of this technology in manipulating neuronal activity and for high-efficiency sparse labeling of neuronal populations.

    更新日期:2017-11-16
  • Maternal Gdf3 is an obligatory cofactor in nodal signaling for embryonic axis formation in zebrafish
    eLife (IF 7.725) Pub Date : 2017-11-15
    Brent W Bisgrove, Yi-Chu Su, H Joseph Yost

    Zebrafish Gdf3 (Dvr1) is a member of the TGFb superfamily of cell signaling ligands that includes Xenopus Vg1 and mammalian Gdf1/3. Surprisingly, engineered homozygous mutants in zebrafish have no apparent phenotype. Elimination of Gdf3 in oocytes of maternal-zygotic mutants results in embryonic lethality that can be fully rescued with gdf3 RNA, demonstrating that Gdf3 is required only early in development, beyond which mutants are viable and fertile. Gdf3 mutants are refractory to Nodal ligands and Nodal repressor Lefty1. Signaling driven by TGFb ligand Activin and constitutively active receptors Alk4 and Alk2 remain intact in gdf3 mutants, indicating that Gdf3 functions at the same pathway step as Nodal. Targeting gdf3 and ndr2 RNA to specific lineages indicates that exogenous gdf3 is able to fully rescue mutants only when co-expressed with endogenous Nodal. Together, these findings demonstrate that Gdf3 is an essential cofactor of Nodal signaling during establishment of the embryonic axis.

    更新日期:2017-11-16
  • Bladder cancer associated mutations in RXRA activate peroxisome proliferator-activated receptors to drive urothelial Proliferation
    eLife (IF 7.725) Pub Date : 2017-11-16
    Angela M Halstead, Chiraag D Kapadia, Jennifer Bolzenius, Clarence E Chu, Andrew Schriefer, Lukas D Wartman, Gregory R Bowman, Vivek K Arora

    RXRA regulates transcription as part of a heterodimer with 14 other nuclear receptors, including the peroxisome proliferator-activated receptors (PPARs). Analysis from the TCGA raised the possibility that hyperactive PPAR signaling, either due to PPAR gamma gene amplification or RXRA hot-spot mutation (S427F/Y) drives 20-25% of human bladder cancers. Here we characterize mutant RXRA, demonstrating it induces enhancer/promoter activity in the context of RXRA/PPAR heterodimers in human bladder cancer cells. Structure-function studies indicate that the RXRA substitution allosterically regulates the PPAR AF2 domain via an aromatic interaction with the terminal tyrosine found in PPARs. In mouse urothelial organoids, PPAR agonism is sufficient to drive growth-factor independent growth in the context of concurrent tumor suppressor loss. Similarly, mutant RXRA stimulates growth-factor independent growth of Trp53/Kdm6a null bladder organoids. Mutant RXRA driven growth of urothelium is reversible by PPAR inhibition, supporting PPARs as targetable drivers of bladder cancer.

    更新日期:2017-11-16
  • Structural basis of host recognition and biofilm formation by Salmonella Saf pili
    eLife (IF 7.725) Pub Date : 2017-11-10
    Longhui Zeng, Li Zhang, Pengran Wang, Guoyu Meng

    Pili are critical in host recognition, colonization and biofilm formation during bacterial infection. Here, we report the crystal structures of SafD-dsc and SafD-SafA-SafA (SafDAA-dsc) in Saf pili. Cell adherence assays show that SafD and SafA are both required for host recognition, suggesting a poly-adhesive mechanism for Saf pili. Moreover, the SafDAA-dsc structure, as well as SAXS characterization, reveals an unexpected inter-molecular oligomerization, prompting the investigation of Saf-driven self-association in biofilm formation. The bead/cell aggregation and biofilm formation assays are used to demonstrate the novel function of Saf pili. Structure-based mutants targeting the inter-molecular hydrogen bonds and complementary architecture/surfaces in SafDAA-dsc dimers significantly impaired the Saf self-association activity and biofilm formation. In summary, our results identify two novel functions of Saf pili: the poly-adhesive and self-associating activities. More importantly, Saf-Saf structures and functional characterizations help to define a pili-mediated inter-cellular oligomerizaiton mechanism for bacterial aggregation, colonization and ultimate biofilm formation.

    更新日期:2017-11-16
  • Pharmacological augmentation of nicotinamide phosphoribosyltransferase (NAMPT) protects against paclitaxel-induced peripheral neuropathy
    eLife (IF 7.725) Pub Date : 2017-11-10
    Peter M LoCoco, April L Risinger, Hudson R Smith, Teresa S Chavera, Kelly A Berg, William P Clarke

    Chemotherapy-induced peripheral neuropathy (CIPN) arises from collateral damage to peripheral afferent sensory neurons by anticancer pharmacotherapy, leading to debilitating neuropathic pain. No effective treatment for CIPN exists, short of dose-reduction which worsens cancer prognosis. Here we report that stimulation of nicotinamide phosphoribosyltransferase (NAMPT) produced robust neuroprotection in an aggressive CIPN model utilizing the frontline anticancer drug, paclitaxel (PTX). Daily treatment of rats with the first-in-class NAMPT stimulator, P7C3-A20, prevented behavioral and histologic indicators of peripheral neuropathy, stimulated tissue NAD recovery, improved general health, and abolished attrition produced by a near maximum-tolerated dose of PTX. Inhibition of NAMPT blocked P7C3-A20-mediated neuroprotection, whereas supplementation with the NAMPT substrate, nicotinamide, potentiated a subthreshold dose of P7C3-A20 to full efficacy. Importantly, P7C3-A20 blocked PTX-induced allodynia in tumored mice without reducing antitumoral efficacy. These findings identify enhancement of NAMPT activity as a promising new therapeutic strategy to protect against anticancer drug-induced peripheral neurotoxicity.

    更新日期:2017-11-16
  • Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis
    eLife (IF 7.725) Pub Date : 2017-11-10
    Martin Steger, Federico Diez, Herschel S Dhekne, Pawel Lis, Raja S Nirujogi, Ozge Karayel, Francesca Tonelli, Terina N Martinez, Esben Lorentzen, Suzanne R Pfeffer, Dario R Alessi, Matthias Mann

    We previously reported that Parkinson's disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger, Tonelli et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

    更新日期:2017-11-16
  • Regulatory network structure determines patterns of intermolecular epistasis
    eLife (IF 7.725) Pub Date : 2017-11-13
    Mato Lagator, Srdjan Sarikas, Hande Acar, Jonathan P Bollback, Călin C Guet

    Most phenotypes are determined by molecular systems composed of specifically interacting molecules. However, unlike for individual components, little is known about the distributions of mutational effects of molecular systems as a whole. We ask how the distribution of mutational effects of a transcriptional regulatory system differs from the distributions of its components, by first independently, and then simultaneously, mutating a transcription factor and the associated promoter it represses. We find that the system distribution exhibits increased phenotypic variation compared to individual component distributions - an effect arising from intermolecular epistasis between the transcription factor and its DNA-binding site. In large part, this epistasis can be qualitatively attributed to the structure of the transcriptional regulatory system, and could therefore be a common feature in prokaryotes. Counter-intuitively, intermolecular epistasis can alleviate the constraints of individual components, thereby increasing phenotypic variation that selection could act on and facilitating adaptive evolution.

    更新日期:2017-11-16
  • Ubiquitin turnover and endocytic trafficking in yeast are regulated by Ser57 phosphorylation of ubiquitin
    eLife (IF 7.725) Pub Date : 2017-11-13
    Sora Lee, Jessica M Tumolo, Aaron C Ehlinger, Kristin K Jernigan, Susan J Qualls-Histed, Pi-Chiang Hsu, W Hayes McDonald, Walter J Chazin, Jason A MacGurn

    Despite its central role in protein degradation little is known about the molecular mechanisms that sense, maintain, and regulate steady state concentration of ubiquitin in the cell. Here, we describe a novel mechanism for regulation of ubiquitin homeostasis that is mediated by phosphorylation of ubiquitin at the Ser57 position. We find that loss of Ppz phosphatase activity leads to defects in ubiquitin homeostasis that are at least partially attributable to elevated levels of Ser57 phosphorylated ubiquitin. Phosphomimetic mutation at the Ser57 position of ubiquitin conferred increased rates of endocytic trafficking and ubiquitin turnover. These phenotypes are associated with bypass of recognition by endosome-localized deubiquitylases - including Doa4 which is critical for regulation of ubiquitin recycling. Thus, ubiquitin homeostasis is significantly impacted by the rate of ubiquitin flux through the endocytic pathway and by signaling pathways that converge on ubiquitin itself to determine whether it is recycled or degraded in the vacuole.

    更新日期:2017-11-16
  • Simultaneous enumeration of cancer and immune cell types from bulk tumor gene expression data
    eLife (IF 7.725) Pub Date : 2017-11-13
    Julien Racle, Kaat de Jonge, Petra Baumgaertner, Daniel E Speiser, David Gfeller

    Immune cells infiltrating tumors can have important impact on tumor progression and response to therapy. We present an efficient algorithm to simultaneously estimate the fraction of cancer and immune cell types from bulk tumor gene expression data. Our method integrates novel gene expression profiles from each major non-malignant cell type found in tumors, renormalization based on cell-type specific mRNA content, and the ability to consider uncharacterized and possibly highly variable cell types. Feasibility is demonstrated by validation with flow cytometry, immunohistochemistry and single-cell RNA-Seq analyses of human melanoma and colorectal tumor specimens. Altogether, our work not only improves accuracy but also broadens the scope of absolute cell fraction predictions from tumor gene expression data, and provides a unique novel experimental benchmark for immunogenomics analyses in cancer research.

    更新日期:2017-11-16
  • Point of View: A transatlantic perspective on 20 emerging issues in biological engineering
    eLife (IF 7.725) Pub Date : 2017-11-14
    Bonnie C Wintle, Christian R Boehm, Catherine Rhodes, Jennifer C Molloy, Piers Millett, Laura Adam, Rainer Breitling, Rob Carlson, Rocco Casagrande, Malcolm Dando, Robert Doubleday, Eric Drexler, Brett Edwards, Tom Ellis, Nicholas G Evans, Richard Hammond, Jim Haseloff, Linda Kahl, Todd Kuiken, Benjamin R Lichman, Colette A Matthewman, Johnathan A Napier, Seán S ÓhÉigeartaigh, Nicola J Patron, Edward Perello, Philip Shapira, Joyce Tait, Eriko Takano, William J Sutherland

    Advances in biological engineering are likely to have substantial impacts on global society. To explore these potential impacts we ran a horizon scanning exercise to capture a range of perspectives on the opportunities and risks presented by biological engineering. We first identified 70 potential issues, and then used an iterative process to prioritise 20 issues that we considered to be emerging, to have potential global impact, and to be relatively unknown outside the field of biological engineering. The issues identified may be of interest to researchers, businesses and policy makers in sectors such as health, energy, agriculture and the environment. https://doi.org/10.7554/eLife.30247.001

    更新日期:2017-11-16
  • Defective synaptic transmission causes disease signs in a mouse model of Juvenile Neuronal Ceroid Lipofuscinosis
    eLife (IF 7.725) Pub Date : 2017-11-14
    Benedikt Grünewald, Maren D Lange, Christian Werner, Aet O'Leary, Andreas Weishaupt, Sandy Popp, David A Pearce, Heinz Wiendl, Andreas Reif, Hans C Pape, Klaus V Toyka, Claudia Sommer, Christian Geis

    Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease) caused by mutations in the CLN3 gene is the most prevalent inherited neurodegenerative disease in childhood resulting in widespread central nervous system dysfunction and premature death. The consequences of CLN3 mutation on the progression of the disease, on neuronal transmission, and on central nervous network dysfunction are poorly understood. We used Cln3 knockout (Cln3Δex7/8) mice and found increased anxiety-related behavior and impaired aversive learning as well as markedly affected motor function including disordered coordination. Patch-clamp and loose-patch recordings revealed severely affected inhibitory and excitatory synaptic transmission in amygdala, hippocampus, and in cerebellar networks. Changes in presynaptic release properties may result from dysfunction of CLN3 protein. Furthermore, loss of calbindin, neuropeptide Y, parvalbumin, and GAD65-positive interneurons in central networks collectively support the hypothesis that degeneration of GABAergic interneurons may be the cause of supraspinal GABAergic disinhibition.

    更新日期:2017-11-16
  • Distinct spatial coordinate of visual and vestibular heading signals in macaque FEFsem and MSTd
    eLife (IF 7.725) Pub Date : 2017-11-14
    Lihua Yang, Yong Gu

    Precise heading estimate requires integration of visual optic flow and vestibular inertial motion originating from distinct spatial coordinates (eye- and head-centered, respectively). To explore whether the two heading signals may share a common reference frame along the hierarchy of cortical stages, we explored two multisensory areas in macaques: the smooth pursuit area of the frontal eye field (FEFsem) closer to the motor side, and the dorsal portion of medial superior temporal area (MSTd) closer to the sensory side. In both areas, vestibular signals are head-centered, whereas visual signals are mainly eye-centered. However, visual signals in FEFsem are more shifted towards the head coordinate compared to MSTd. These results are robust being largely independent on: (1) smooth pursuit eye movement, (2) motion parallax cue, and (3) behavioral context for active heading estimation, indicating that the visual and vestibular heading signals may be represented in distinct spatial coordinate in sensory cortices. https://doi.org/10.7554/eLife.29809.001

    更新日期:2017-11-16
  • Gyrification of the cerebral cortex requires FGF signaling in the mammalian brain
    eLife (IF 7.725) Pub Date : 2017-11-14
    Naoyuki Matsumoto, Yohei Shinmyo, Yoshie Ichikawa, Hiroshi Kawasaki

    Although it has been believed that the evolution of cortical folds was a milestone, allowing for an increase in the number of neurons in the cerebral cortex, the mechanisms underlying the formation of cortical folds are largely unknown. Here we show regional differences in the expression of fibroblast growth factor receptors (FGFRs) in the developing cerebral cortex of ferrets even before cortical folds are formed. By taking the advantage of our in utero electroporation technique for ferrets, we found that cortical folding was impaired in the ferret cerebral cortex when FGF signaling was inhibited. We also found that FGF signaling was crucial for producing Pax6-positive neural progenitors in the outer subventricular zone (OSVZ) of the developing cerebral cortex. Furthermore, we found that upper layers of the cerebral cortex were preferentially reduced by inhibiting FGF signaling. Our results shed light on the mechanisms of cortical folding in gyrencephalic mammalian brains. https://doi.org/10.7554/eLife.29285.001

    更新日期:2017-11-16
  • An adaptation of astronomical image processing enables characterization and functional 3D mapping of individual sites of excitation-contraction coupling in rat cardiac muscle
    eLife (IF 7.725) Pub Date : 2017-11-14
    Qinghai Tian, Lars Kaestner, Laura Schröder, Jia Guo, Peter Lipp

    In beating cardiomyocytes, synchronized localized Ca2+ transients from thousands of active excitation-contraction coupling sites (ECC couplons) comprising plasma and sarcoplasmic reticulum membrane calcium channels are important determinants of the heart's performance. Nevertheless, our knowledge about their properties is limited by the lack of appropriate experimental and analysis strategies. We designed CaCLEAN to untangle fundamental characteristics of ECC couplons by combining the astronomer's CLEAN algorithm with known properties of calcium diffusion. CaCLEAN empowers the investigation of fundamental properties of ECC couplons in beating cardiomyocytes without pharmacological interventions. On the nanoscopic level of individual ECC couplons, we reveal their role in the negative amplitude-frequency relationship and b-adrenergic stimulation, including decreasing and increasing firing reliability, respectively. CaCLEAN combined with 3D confocal imaging of beating cardiomyocytes provides a functional 3D map of active ECC couplons (on average 17.000 per myocyte). CaCLEAN will further enlighten remodelling processes of ECC couplons underlying cardiac diseases.

    更新日期:2017-11-16
  • Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals
    eLife (IF 7.725) Pub Date : 2017-11-14
    Inge Kühl, Maria Miranda, Ilian Atanassov, Irina Kuznetsova, Yvonne Hinze, Arnaud Mourier, Aleksandra Filipovska, Nils-Göran Larsson

    Dysfunction of the oxidative phosphorylation (OXPHOS) system is a major cause of human disease and the cellular consequences are highly complex. Here, we present comparative analyses of mitochondrial proteomes, cellular transcriptomes and targeted metabolomics of five knockout mouse strains deficient in essential factors required for mitochondrial DNA gene expression, leading to OXPHOS dysfunction. Moreover, we describe sequential protein changes during post-natal development and progressive OXPHOS dysfunction in time course analyses in control mice and a middle lifespan knockout, respectively. Very unexpectedly, we identify a new response pathway to OXPHOS dysfunction in which the intra-mitochondrial synthesis of coenzyme Q (ubiquinone, Q) and Q levels are profoundly decreased, pointing towards novel possibilities for therapy. Our extensive omics analyses provide a high-quality resource of altered gene expression patterns under severe OXPHOS deficiency comparing several mouse models, that will deepen our understanding, open avenues for research and provide an important reference for diagnosis and treatment.

    更新日期:2017-11-16
  • Subcellular analysis of pigeon hair cells implicates vesicular trafficking in cuticulosome formation and maintenance
    eLife (IF 7.725) Pub Date : 2017-11-15
    Simon Nimpf, Erich Pascal Malkemper, Mattias Lauwers, Lyubov Ushakova, Gregory Nordmann, Andrea Wenninger-Weinzierl, Thomas R Burkard, Sonja Jacob, Thomas Heuser, Guenter P Resch, David A Keays

    Hair cells are specialized sensors located in the inner ear that enable the transduction of sound, motion, and gravity into neuronal impulses. In birds some hair cells contain an iron-rich organelle, the cuticulosome, that has been implicated in the magnetic sense. Here, we exploit histological, transcriptomic and tomographic methods to investigate the development of cuticulosomes, as well as the molecular and subcellular architecture of cuticulosome positive hair cells. We show that this organelle forms rapidly after hatching in a process that involves vesicle fusion and nucleation of ferritin nanoparticles. We further report that transcripts involved in endocytosis, extracellular exosomes, and metal ion binding are differentially expressed in cuticulosome positive hair cells. These data suggest that the cuticulosome and the associated molecular machinery regulate the concentration of iron within the labyrinth of the inner ear, which might indirectly tune a magnetic sensor that relies on electromagnetic induction.

    更新日期:2017-11-16
  • Maternal Gdf3 is an obligatory cofactor in nodal signaling for embryonic axis formation in zebrafish
    eLife (IF 7.725) Pub Date : 2017-11-15
    Brent W Bisgrove, Yi-Chu Su, H Joseph Yost

    Zebrafish Gdf3 (Dvr1) is a member of the TGFb superfamily of cell signaling ligands that includes Xenopus Vg1 and mammalian Gdf1/3. Surprisingly, engineered homozygous mutants in zebrafish have no apparent phenotype. Elimination of Gdf3 in oocytes of maternal-zygotic mutants results in embryonic lethality that can be fully rescued with gdf3 RNA, demonstrating that Gdf3 is required only early in development, beyond which mutants are viable and fertile. Gdf3 mutants are refractory to Nodal ligands and Nodal repressor Lefty1. Signaling driven by TGFb ligand Activin and constitutively active receptors Alk4 and Alk2 remain intact in gdf3 mutants, indicating that Gdf3 functions at the same pathway step as Nodal. Targeting gdf3 and ndr2 RNA to specific lineages indicates that exogenous gdf3 is able to fully rescue mutants only when co-expressed with endogenous Nodal. Together, these findings demonstrate that Gdf3 is an essential cofactor of Nodal signaling during establishment of the embryonic axis.

    更新日期:2017-11-16
  • DDM1 and Lsh remodelers allow methylation of DNA wrapped in nucleosomes
    eLife (IF 7.725) Pub Date : 2017-11-15
    David B Lyons, Daniel Zilberman

    Cytosine methylation regulates essential genome functions across eukaryotes, but the fundamental question of whether nucleosomal or naked DNA is the preferred substrate of plant and animal methyltransferases remains unresolved. Here, we show that genetic inactivation of a single DDM1/Lsh family nucleosome remodeler biases methylation toward inter-nucleosomal linker DNA in Arabidopsis thaliana and mouse. We find that DDM1 enables methylation of DNA bound to the nucleosome, suggesting that nucleosome-free DNA is the preferred substrate of eukaryotic methyltransferases in vivo. Furthermore, we show that simultaneous mutation of DDM1 and linker histone H1 in Arabidopsis reproduces the strong linker-specific methylation patterns of species that diverged from flowering plants and animals over a billion years ago. Our results indicate that in the absence of remodeling, nucleosomes are strong barriers to DNA methyltransferases. Linker-specific methylation can evolve simply by breaking the connection between nucleosome remodeling and DNA methylation.

    更新日期:2017-11-16
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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