Cell

A C-Type Lectin Collaborates with a CD45 Phosphatase Homolog to Facilitate West Nile Virus Infection of Mosquitoes.

Publication Date: 2010 Aug 25 PMID: 20797779
Authors: Cheng, G. - Cox, J. - Wang, P. - Krishnan, M. N. - Dai, J. - Qian, F. - Anderson, J. F. - Fikrig, E.
Journal: Cell

West Nile virus (WNV) is the most common arthropod-borne flavivirus in the United States; however, the vector ligand(s) that participate in infection are not known. We now show that an Aedes aegypti C-type lectin, mosGCTL-1, is induced by WNV, interacts with WNV in a calcium-dependent manner, and facilitates infection in vivo and in vitro. A mosquito homolog of human CD45 in A. aegypti, designated mosPTP-1, recruits mosGCTL-1 to enable viral attachment to cells and to enhance viral entry. In vivo experiments show that mosGCTL-1 and mosPTP-1 function as part of the same pathway and are critical for WNV infection of mosquitoes. A similar phenomenon was also observed in Culex quinquefasciatus, a natural vector of WNV, further demonstrating that these genes participate in WNV infection. During the mosquito blood-feeding process, WNV infection was blocked in vivo with mosGCTL-1 antibodies. A molecular understanding of flaviviral-arthropod interactions may lead to strategies to control viral dissemination in nature.

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Structural Basis of Semaphorin-Plexin Recognition and Viral Mimicry from Sema7A and A39R Complexes with PlexinC1.

Publication Date: 2010 Aug 18 PMID: 20727575
Authors: Liu, H. - Juo, Z. S. - Shim, A. H. - Focia, P. J. - Chen, X. - Garcia, K. C. - He, X.
Journal: Cell

Repulsive signaling by Semaphorins and Plexins is crucial for the development and homeostasis of the nervous, immune, and cardiovascular systems. Sema7A acts as both an immune and a neural Semaphorin through PlexinC1, and A39R is a Sema7A mimic secreted by smallpox virus. We report the structures of Sema7A and A39R complexed with the Semaphorin-binding module of PlexinC1. Both structures show two PlexinC1 molecules symmetrically bridged by Semaphorin dimers, in which the Semaphorin and PlexinC1 beta propellers interact in an edge-on, orthogonal orientation. Both binding interfaces are dominated by the insertion of the Semaphorin's 4c-4d loop into a deep groove in blade 3 of the PlexinC1 propeller. A39R appears to achieve Sema7A mimicry by preserving key Plexin-binding determinants seen in the mammalian Sema7A complex that have evolved to achieve higher affinity binding to the host-derived PlexinC1. The complex structures support a conserved Semaphorin-Plexin recognition mode and suggest that Plexins are activated by dimerization.

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SnapShot: Homologous recombination in DNA double-strand break repair.

Publication Date: 2010 Aug 20 PMID: 20723763
Authors: Mazon, G. - Mimitou, E. P. - Symington, L. S.
Journal: Cell

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Activation of specific apoptotic caspases with an engineered small-molecule-activated protease.

Publication Date: 2010 Aug 20 PMID: 20723762
Authors: Gray, D. C. - Mahrus, S. - Wells, J. A.
Journal: Cell

Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-tobacco etch virus (TEV) protease under small-molecule control, which we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.

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Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress.

Publication Date: 2010 Aug 20 PMID: 20723761
Authors: Seifert, U. - Bialy, L. P. - Ebstein, F. - Bech-Otschir, D. - Voigt, A. - Schroter, F. - Prozorovski, T. - Lange, N. - Steffen, J. - Rieger, M. - Kuckelkorn, U. - Aktas, O. - Kloetzel, P. M. - Kruger, E.
Journal: Cell

Interferon (IFN)-induced immunoproteasomes (i-proteasomes) have been associated with improved processing of major histocompatibility complex (MHC) class I antigens. Here, we show that i-proteasomes function to protect cell viability under conditions of IFN-induced oxidative stress. IFNs trigger the production of reactive oxygen species, which induce protein oxidation and the formation of nascent, oxidant-damaged proteins. We find that the ubiquitylation machinery is concomitantly upregulated in response to IFNs, functioning to target defective ribosomal products (DRiPs) for degradation by i-proteasomes. i-proteasome-deficiency in cells and in murine inflammation models results in the formation of aggresome-like induced structures and increased sensitivity to apoptosis. Efficient clearance of these aggregates by the enhanced proteolytic activity of the i-proteasome is important for the preservation of cell viability upon IFN-induced oxidative stress. Our findings suggest that rather than having a specific role in the production of class I antigens, i-proteasomes increase the peptide supply for antigen presentation as part of a more general role in the maintenance of protein homeostasis.

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Identification of MOAG-4/SERF as a regulator of age-related proteotoxicity.

Publication Date: 2010 Aug 20 PMID: 20723760
Authors: van Ham, T. J. - Holmberg, M. A. - van der Goot, A. T. - Teuling, E. - Garcia-Arencibia, M. - Kim, H. E. - Du, D. - Thijssen, K. L. - Wiersma, M. - Burggraaff, R. - van Bergeijk, P. - van Rheenen, J. - Jerre van Veluw, G. - Hofstra, R. M. - Rubinsztein, D. C. - Nollen, E. A.
Journal: Cell

Fibrillar protein aggregates are the major pathological hallmark of several incurable, age-related, neurodegenerative disorders. These aggregates typically contain aggregation-prone pathogenic proteins, such as amyloid-beta in Alzheimer's disease and alpha-synuclein in Parkinson's disease. It is, however, poorly understood how these aggregates are formed during cellular aging. Here we identify an evolutionarily highly conserved modifier of aggregation, MOAG-4, as a positive regulator of aggregate formation in C. elegans models for polyglutamine diseases. Inactivation of MOAG-4 suppresses the formation of compact polyglutamine aggregation intermediates that are required for aggregate formation. The role of MOAG-4 in driving aggregation extends to amyloid-beta and alpha-synuclein and is evolutionarily conserved in its human orthologs SERF1A and SERF2. MOAG-4/SERF appears to act independently from HSF-1-induced molecular chaperones, proteasomal degradation, and autophagy. Our results suggest that MOAG-4/SERF regulates age-related proteotoxicity through a previously unexplored pathway, which will open up new avenues for research on age-related, neurodegenerative diseases.

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ATG12 conjugation to ATG3 regulates mitochondrial homeostasis and cell death.

Publication Date: 2010 Aug 20 PMID: 20723759
Authors: Radoshevich, L. - Murrow, L. - Chen, N. - Fernandez, E. - Roy, S. - Fung, C. - Debnath, J.
Journal: Cell

ATG12, an ubiquitin-like modifier required for macroautophagy, has a single known conjugation target, another autophagy regulator called ATG5. Here, we identify ATG3 as a substrate for ATG12 conjugation. ATG3 is the E2-like enzyme necessary for ATG8/LC3 lipidation during autophagy. ATG12-ATG3 complex formation requires ATG7 as the E1 enzyme and ATG3 autocatalytic activity as the E2, resulting in the covalent linkage of ATG12 onto a single lysine on ATG3. Surprisingly, disrupting ATG12 conjugation to ATG3 does not affect starvation-induced autophagy. Rather, the lack of ATG12-ATG3 complex formation produces an expansion in mitochondrial mass and inhibits cell death mediated by mitochondrial pathways. Overall, these results unveil a role for ATG12-ATG3 in mitochondrial homeostasis and implicate the ATG12 conjugation system in cellular functions distinct from the early steps of autophagosome formation.

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Structural basis for negative cooperativity in growth factor binding to an EGF receptor.

Publication Date: 2010 Aug 20 PMID: 20723758
Authors: Alvarado, D. - Klein, D. E. - Lemmon, M. A.
Journal: Cell

Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the "high-affinity" and "low-affinity" classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

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The monopolin complex crosslinks kinetochore components to regulate chromosome-microtubule attachments.

Publication Date: 2010 Aug 20 PMID: 20723757
Authors: Corbett, K. D. - Yip, C. K. - Ee, L. S. - Walz, T. - Amon, A. - Harrison, S. C.
Journal: Cell

The monopolin complex regulates different types of kinetochore-microtubule attachments in fungi, ensuring sister chromatid co-orientation in Saccharomyces cerevisiae meiosis I and inhibiting merotelic attachment in Schizosaccharomyces pombe mitosis. In addition, the monopolin complex maintains the integrity and silencing of ribosomal DNA (rDNA) repeats in the nucleolus. We show here that the S. cerevisiae Csm1/Lrs4 monopolin subcomplex has a distinctive V-shaped structure, with two pairs of protein-protein interaction domains positioned approximately 10 nm apart. Csm1 presents a conserved hydrophobic surface patch that binds two kinetochore proteins: Dsn1, a subunit of the outer-kinetochore MIND/Mis12 complex, and Mif2/CENP-C. Csm1 point-mutations that disrupt kinetochore-subunit binding also disrupt sister chromatid co-orientation in S. cerevisiae meiosis I. We further show that the same Csm1 point-mutations affect rDNA silencing, probably by disrupting binding to the rDNA-associated protein Tof2. We propose that Csm1/Lrs4 functions as a molecular clamp, crosslinking kinetochore components to enforce sister chromatid co-orientation in S. cerevisiae meiosis I and to suppress merotelic attachment in S. pombe mitosis, and crosslinking rDNA repeats to aid rDNA silencing.

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PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps.

Publication Date: 2010 Aug 20 PMID: 20723756
Authors: Park, J. - Myong, S. - Niedziela-Majka, A. - Lee, K. S. - Yu, J. - Lohman, T. M. - Ha, T.
Journal: Cell

Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA-bound proteins, and basic properties like the elementary step size remain controversial. Using single-molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a single-stranded loop. Static disorder limited previous ensemble studies of a PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 nt. This reeling-in activity requires the open conformation of PcrA and can rapidly dismantle a preformed RecA filament even at low PcrA concentrations, suggesting a mode of action for eliminating potentially deleterious recombination intermediates.

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Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival.

Publication Date: 2010 Aug 20 PMID: 20723755
Authors: Zhou, X. - Wang, J. L. - Lu, J. - Song, Y. - Kwak, K. S. - Jiao, Q. - Rosenfeld, R. - Chen, Q. - Boone, T. - Simonet, W. S. - Lacey, D. L. - Goldberg, A. L. - Han, H. Q.
Journal: Cell

Muscle wasting and cachexia have long been postulated to be key determinants of cancer-related death, but there has been no direct experimental evidence to substantiate this hypothesis. Here, we show that in several cancer cachexia models, pharmacological blockade of ActRIIB pathway not only prevents further muscle wasting but also completely reverses prior loss of skeletal muscle and cancer-induced cardiac atrophy. This treatment dramatically prolongs survival, even of animals in which tumor growth is not inhibited and fat loss and production of proinflammatory cytokines are not reduced. ActRIIB pathway blockade abolished the activation of the ubiquitin-proteasome system and the induction of atrophy-specific ubiquitin ligases in muscles and also markedly stimulated muscle stem cell growth. These findings establish a crucial link between activation of the ActRIIB pathway and the development of cancer cachexia. Thus ActRIIB antagonism is a promising new approach for treating cancer cachexia, whose inhibition per se prolongs survival.

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Cellular strategies for regulating DNA supercoiling: a single-molecule perspective.

Publication Date: 2010 Aug 20 PMID: 20723754
Authors: Koster, D. A. - Crut, A. - Shuman, S. - Bjornsti, M. A. - Dekker, N. H.
Journal: Cell

Entangling and twisting of cellular DNA (i.e., supercoiling) are problems inherent to the helical structure of double-stranded DNA. Supercoiling affects transcription, DNA replication, and chromosomal segregation. Consequently the cell must fine-tune supercoiling to optimize these key processes. Here, we summarize how supercoiling is generated and review experimental and theoretical insights into supercoil relaxation. We distinguish between the passive dissipation of supercoils by diffusion and the active removal of supercoils by topoisomerase enzymes. We also review single-molecule studies that elucidate the timescales and mechanisms of supercoil removal.

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The Immunoproteasome Cleans up after Inflammation.

Publication Date: 2010 Aug 20 PMID: 20723753
Authors: van Deventer, S. - Neefjes, J.
Journal: Cell

Immune cells and cells activated by the inflammatory cytokine interferon express variant proteasomes called immunoproteasomes that are characterized by unique catalytic subunits. Seifert et al. (2010) now show in mouse models of inflammatory disease that immunoproteasomes help prevent the accumulation of harmful protein aggregates.

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Up a hydrophobic creek with a short paddle.

Publication Date: 2010 Aug 20 PMID: 20723752
Authors: Hulse, R. E. - Li, Q. - Perozo, E.
Journal: Cell

The mechanism by which voltage-dependent ion channels sense membrane potentials has been the most intensively studied and debated topic in modern ion channel research. In this issue, Xu et al. (2010) provide new insights into the minimal topological and physicochemical features required for voltage sensing.

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The ins and outs of EGFR asymmetry.

Publication Date: 2010 Aug 20 PMID: 20723751
Authors: Leahy, D. J.
Journal: Cell

The epidermal growth factor receptor (EGFR) regulates cell proliferation in many tissues. A new structure of the Drosophila EGFR presented by Alvarado et al. (2010) reveals an asymmetric dimer with the ligand bound to only one subunit. The structure provides a rationale for the receptor's negative cooperativity and necessitates a reconsideration of models for activation of human EGFR.

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Reversing cachexia.

Publication Date: 2010 Aug 20 PMID: 20723750
Authors: Tisdale, M. J.
Journal: Cell

Muscle atrophy (cachexia) in cancer patients is a life-threatening condition for which therapeutic options are limited. Zhou et al. (2010) now identify a new target for treating cachexia, the activin type-2 receptor (ActRIIB). In several mouse models of cachexia, the authors reversed wasting of skeletal and cardiac muscle and increased life span by blocking ActRIIB with a decoy receptor.

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Induced domain formation in endocytic invagination, lipid sorting, and scission.

Publication Date: 2010 Aug 20 PMID: 20723749
Authors: Johannes, L. - Mayor, S.
Journal: Cell

Lipid segregation occurs in biological membranes, but how this plays into cellular processes like endocytosis has been unclear. Here, we discuss how the active or passive induction of lipid-protein domain formation in membranes can alter membrane mechanics and thus affect processes such as the generation of curvature, the scission of buds and tubules, and lipid sorting.

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An alternative splicing network links cell-cycle control to apoptosis.

Publication Date: 2010 Aug 20 PMID: 20705336
Authors: Moore, M. J. - Wang, Q. - Kennedy, C. J. - Silver, P. A.
Journal: Cell

Alternative splicing is a vast source of biological regulation and diversity that is misregulated in cancer and other diseases. To investigate global control of alternative splicing in human cells, we analyzed splicing of mRNAs encoding Bcl2 family apoptosis factors in a genome-wide siRNA screen. The screen identified many regulators of Bcl-x and Mcl1 splicing, notably an extensive network of cell-cycle factors linked to aurora kinase A. Drugs or siRNAs that induce mitotic arrest promote proapoptotic splicing of Bcl-x, Mcl1, and caspase-9 and alter splicing of other apoptotic transcripts. This response precedes mitotic arrest, indicating coordinated upregulation of prodeath splice variants that promotes apoptosis in arrested cells. These shifts correspond to posttranslational turnover of splicing regulator ASF/SF2, which directly binds and regulates these target mRNAs and globally regulates apoptosis. Broadly, our results reveal an alternative splicing network linking cell-cycle control to apoptosis.

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SnapShot: Nonhomologous DNA end joining (NHEJ).

Publication Date: 2010 Aug 6 PMID: 20691907
Authors: Lieber, M. R. - Wilson, T. E.
Journal: Cell

MeSH Categories: Animals, DNA/metabolism, DNA Breaks, Double-Stranded, *DNA Repair, DNA Repair Enzymes/metabolism, Humans

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Myc-nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation.

Publication Date: 2010 Aug 6 PMID: 20691906
Authors: Conacci-Sorrell, M. - Ngouenet, C. - Eisenman, R. N.
Journal: Cell

The Myc oncoprotein family comprises transcription factors that control multiple cellular functions and are widely involved in oncogenesis. Here we report the identification of Myc-nick, a cytoplasmic form of Myc generated by calpain-dependent proteolysis at lysine 298 of full-length Myc. Myc-nick retains conserved Myc box regions but lacks nuclear localization signals and the bHLHZ domain essential for heterodimerization with Max and DNA binding. Myc-nick induces alpha-tubulin acetylation and altered cell morphology by recruiting histone acetyltransferase GCN5 to microtubules. During muscle differentiation, while the levels of full-length Myc diminish, Myc-nick and acetylated alpha-tubulin levels are increased. Ectopic expression of Myc-nick accelerates myoblast fusion, triggers the expression of myogenic markers, and permits Myc-deficient fibroblasts to transdifferentiate in response to MyoD. We propose that the cleavage of Myc by calpain abrogates the transcriptional inhibition of differentiation by full-length Myc and generates Myc-nick, a driver of cytoplasmic reorganization and differentiation.

MeSH Categories: Acetylation, Animals, Cell Differentiation, Cell Line, Tumor, Cells, Cultured, Cytosol/metabolism, Humans, Mice, Proto-Oncogene Proteins c-myc/*metabolism, Rats, Tubulin/*metabolism

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Plzf regulates germline progenitor self-renewal by opposing mTORC1.

Publication Date: 2010 Aug 6 PMID: 20691905
Authors: Hobbs, R. M. - Seandel, M. - Falciatori, I. - Rafii, S. - Pandolfi, P. P.
Journal: Cell

Hyperactivity of mTORC1, a key mediator of cell growth, leads to stem cell depletion, although the underlying mechanisms are poorly defined. Using spermatogonial progenitor cells (SPCs) as a model system, we show that mTORC1 impairs stem cell maintenance by a negative feedback from mTORC1 to receptors required to transduce niche-derived signals. We find that SPCs lacking Plzf, a transcription factor essential for SPC maintenance, have enhanced mTORC1 activity. Aberrant mTORC1 activation in Plzf(-/-) SPCs inhibits their response to GDNF, a growth factor critical for SPC self-renewal, via negative feedback at the level of the GDNF receptor. Plzf opposes mTORC1 activity by inducing expression of the mTORC1 inhibitor Redd1. Thus, we identify the mTORC1-Plzf functional interaction as a critical rheostat for maintenance of the spermatogonial pool and propose a model whereby negative feedback from mTORC1 to the GDNF receptor balances SPC growth with self-renewal.

MeSH Categories: Animals, Feedback, Physiological, Glial Cell Line-Derived Neurotrophic Factor/metabolism, Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism, Kruppel-Like Transcription Factors/genetics/*metabolism, Male, Mice, Signal Transduction, Spermatogonia/*cytology/metabolism, Stem Cells/*cytology/metabolism, Testis/cytology, Transcription Factors/*metabolism

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PNPASE regulates RNA import into mitochondria.

Publication Date: 2010 Aug 6 PMID: 20691904
Authors: Wang, G. - Chen, H. W. - Oktay, Y. - Zhang, J. - Allen, E. L. - Smith, G. M. - Fan, K. C. - Hong, J. S. - French, S. W. - McCaffery, J. M. - Lightowlers, R. N. - Morse, H. C. 3rd - Koehler, C. M. - Teitell, M. A.
Journal: Cell

RNA import into mammalian mitochondria is considered essential for replication, transcription, and translation of the mitochondrial genome but the pathway(s) and factors that control this import are poorly understood. Previously, we localized polynucleotide phosphorylase (PNPASE), a 3' --> 5' exoribonuclease and poly-A polymerase, in the mitochondrial intermembrane space, a location lacking resident RNAs. Here, we show a new role for PNPASE in regulating the import of nuclear-encoded RNAs into the mitochondrial matrix. PNPASE reduction impaired mitochondrial RNA processing and polycistronic transcripts accumulated. Augmented import of RNase P, 5S rRNA, and MRP RNAs depended on PNPASE expression and PNPASE-imported RNA interactions were identified. PNPASE RNA processing and import activities were separable and a mitochondrial RNA targeting signal was isolated that enabled RNA import in a PNPASE-dependent manner. Combined, these data strongly support an unanticipated role for PNPASE in mediating the translocation of RNAs into mitochondria.

MeSH Categories: Animals, Cell Line, Gene Knockout Techniques, Humans, Mice, Mice, Inbred C57BL, Mitochondria/*metabolism, Polyribonucleotide Nucleotidyltransferase/genetics/*metabolism, RNA/*metabolism, RNA Processing, Post-Transcriptional, Ribonuclease P/metabolism, Saccharomyces cerevisiae/metabolism

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Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E.

Publication Date: 2010 Aug 6 PMID: 20691903
Authors: Kim, Y. - Holland, A. J. - Lan, W. - Cleveland, D. W.
Journal: Cell

Opposing roles of Aurora kinases and protein phosphatase 1 (PP1) during mitosis have long been suggested. Here, we demonstrate that Aurora kinases A and B phosphorylate a conserved residue on the kinetochore motor CENP-E. PP1 binds CENP-E via a motif overlapping this phosphorylation site and binding is disrupted by Aurora phosphorylation. Phosphorylation of CENP-E by the Auroras is enriched at spindle poles, disrupting binding of PP1 and reducing CENP-E's affinity for individual microtubules. This phosphorylation is required for CENP-E-mediated towing of initially polar chromosomes toward the cell center. Kinetochores on such chromosomes cannot make subsequent stable attachment to spindle microtubules when dephosphorylation of CENP-E or rebinding of PP1 to CENP-E is blocked. Thus, an Aurora/PP1 phosphorylation switch modulates CENP-E motor activity as an essential feature of chromosome congression from poles and localized PP1 delivery by CENP-E to the outer kinetochore is necessary for stable microtubule capture by those chromosomes.

MeSH Categories: Amino Acid Sequence, Animals, Chromosomal Proteins, Non-Histone/*metabolism, Chromosomes/*metabolism, Hela Cells, Humans, Kinetochores/metabolism, Microtubules/metabolism, Mitotic Spindle Apparatus/metabolism, Molecular Sequence Data, Phosphorylation, Protein Phosphatase 1/*metabolism, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases/*metabolism, Sequence Alignment, Xenopus laevis/*metabolism

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Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein.

Publication Date: 2010 Aug 6 PMID: 20691902
Authors: Subramanian, R. - Wilson-Kubalek, E. M. - Arthur, C. P. - Bick, M. J. - Campbell, E. A. - Darst, S. A. - Milligan, R. A. - Kapoor, T. M.
Journal: Cell

Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks are compliant and do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish crosslinks that selectively "mark" antiparallel overlap in dynamic cytoskeletal networks.

MeSH Categories: Cell Cycle Proteins/chemistry/*metabolism, Cryoelectron Microscopy, Humans, Microtubules/*metabolism, Models, Molecular, Protein Structure, Tertiary, Spectrin/metabolism

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A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps.

Publication Date: 2010 Aug 6 PMID: 20691901
Authors: Bieling, P. - Telley, I. A. - Surrey, T.
Journal: Cell

During cell division, microtubules are arranged in a large bipolar structure, the mitotic spindle, to segregate the duplicated chromosomes. Antiparallel microtubule overlaps in the spindle center are essential for establishing bipolarity and maintaining spindle stability throughout mitosis. In anaphase, this antiparallel microtubule array is tightly bundled forming the midzone, which serves as a hub for the recruitment of proteins essential for late mitotic events. The molecular mechanism of midzone formation and the control of its size are not understood. Using an in vitro reconstitution approach, we show here that PRC1 autonomously bundles antiparallel microtubules and recruits Xklp1, a kinesin-4, selectively to overlapping antiparallel microtubules. The processive motor Xklp1 controls overlap size by overlap length-dependent microtubule growth inhibition. Our results mechanistically explain how the two conserved, essential midzone proteins PRC1 and Xklp1 cooperate to constitute a minimal protein module capable of dynamically organizing the core structure of the central anaphase spindle.

MeSH Categories: Anaphase, Animals, Microtubule-Associated Proteins/*metabolism, Microtubules/*metabolism, Mitotic Spindle Apparatus/*metabolism, Tubulin/metabolism, Xenopus Proteins/*metabolism, Xenopus laevis/*metabolism

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Single-stranded DNA transposition is coupled to host replication.

Publication Date: 2010 Aug 6 PMID: 20691900
Authors: Ton-Hoang, B. - Pasternak, C. - Siguier, P. - Guynet, C. - Hickman, A. B. - Dyda, F. - Sommer, S. - Chandler, M.
Journal: Cell

DNA transposition has contributed significantly to evolution of eukaryotes and prokaryotes. Insertion sequences (ISs) are the simplest prokaryotic transposons and are divided into families on the basis of their organization and transposition mechanism. Here, we describe a link between transposition of IS608 and ISDra2, both members of the IS200/IS605 family, which uses obligatory single-stranded DNA intermediates, and the host replication fork. Replication direction through the IS plays a crucial role in excision: activity is maximal when the "top" IS strand is located on the lagging-strand template. Excision is stimulated upon transient inactivation of replicative helicase function or inhibition of Okazaki fragment synthesis. IS608 insertions also exhibit an orientation preference for the lagging-strand template and insertion can be specifically directed to stalled replication forks. An in silico genomic approach provides evidence that dissemination of other IS200/IS605 family members is also linked to host replication.

MeSH Categories: DNA Helicases/metabolism, DNA Primase/metabolism, *DNA Replication, *DNA Transposable Elements, DNA, Single-Stranded/*metabolism, Deinococcus/genetics/*metabolism, Escherichia coli/genetics/*metabolism, Escherichia coli Proteins/metabolism, Trans-Activators/metabolism

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Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.

Publication Date: 2010 Aug 6 PMID: 20691899
Authors: Ieda, M. - Fu, J. D. - Delgado-Olguin, P. - Vedantham, V. - Hayashi, Y. - Bruneau, B. G. - Srivastava, D.
Journal: Cell

The reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) raises the possibility that a somatic cell could be reprogrammed to an alternative differentiated fate without first becoming a stem/progenitor cell. A large pool of fibroblasts exists in the postnatal heart, yet no single "master regulator" of direct cardiac reprogramming has been identified. Here, we report that a combination of three developmental transcription factors (i.e., Gata4, Mef2c, and Tbx5) rapidly and efficiently reprogrammed postnatal cardiac or dermal fibroblasts directly into differentiated cardiomyocyte-like cells. Induced cardiomyocytes expressed cardiac-specific markers, had a global gene expression profile similar to cardiomyocytes, and contracted spontaneously. Fibroblasts transplanted into mouse hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells. We believe these findings demonstrate that functional cardiomyocytes can be directly reprogrammed from differentiated somatic cells by defined factors. Reprogramming of endogenous or explanted fibroblasts might provide a source of cardiomyocytes for regenerative approaches.

MeSH Categories: Animals, *Cell Differentiation, Cell Separation, Fibroblasts/*cytology/metabolism, GATA4 Transcription Factor/metabolism, Gene Expression Profiling, Mice, Muscle Contraction, Myocardium/*cytology, Myocytes, Cardiac/*cytology/metabolism, Myogenic Regulatory Factors/metabolism, T-Box Domain Proteins/metabolism

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The pioneer round of translation: features and functions.

Publication Date: 2010 Aug 6 PMID: 20691898
Authors: Maquat, L. E. - Tarn, W. Y. - Isken, O.
Journal: Cell

In mammalian cells, newly synthesized mRNAs undergo a pioneer round of translation that is important for mRNA quality control. Following maturation of messenger ribonucleoprotein particles during and after the pioneer round, steady-state cycles of mRNA translation generate most of the cell's proteins. Translation factors, RNA-binding proteins, and targets of signaling pathways that are particular to newly synthesized mRNAs regulate critical functions of the pioneer round.

MeSH Categories: Animals, Cell Physiological Phenomena, Humans, *Protein Biosynthesis, RNA Stability, RNA, Messenger/genetics/*metabolism, Ribonucleoproteins/metabolism, Ribosomes/metabolism

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A MAP for bundling microtubules.

Publication Date: 2010 Aug 6 PMID: 20691897
Authors: Walczak, C. E. - Shaw, S. L.
Journal: Cell

Microtubules assemble into arrays of bundled filaments that are critical for multiple steps in cell division, including anaphase and cytokinesis. Recent structural and functional studies, including two papers in this issue of Cell (Bieling et al., 2010; Subramanian et al., 2010), demonstrate how the MAP65 protein PRC1 crosslinks microtubules and cooperates with kinesin motors to control the dynamics and size of bundled regions.

MeSH Categories: Animals, Cell Cycle Proteins/metabolism, Kinesin/metabolism, Microtubule-Associated Proteins/*metabolism, Microtubules/*metabolism, Plants/*metabolism

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Mitochondrial matrix reloaded with RNA.

Publication Date: 2010 Aug 6 PMID: 20691896
Authors: Endo, T. - Yamano, K. - Yoshihisa, T.
Journal: Cell

Although mitochondrial biogenesis requires the import of specific RNAs, the pathways and cellular machineries involved are only poorly understood. Wang et al. (2010) now find that polynucleotide phosphorylase in the intermembrane space of mammalian mitochondria facilitates import of several RNAs into the mitochondrial matrix.

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Stem cells and DNA damage: persist or perish?

Publication Date: 2010 Aug 6 PMID: 20691895
Authors: Lane, A. A. - Scadden, D. T.
Journal: Cell

Stem cells repopulate tissues after injury while also renewing themselves, but this makes them vulnerable to genotoxic damage. Mohrin et al. (2010) and Milyavsky et al. (2010) now show that mouse and human hematopoietic stem cells make opposing decisions about whether to die or to persist in response to DNA damage.

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Noncoding RNAs: the missing "linc" in p53-mediated repression.

Publication Date: 2010 Aug 6 PMID: 20691894
Authors: Barsotti, A. M. - Prives, C.
Journal: Cell

The tumor suppressor protein p53 coordinates the cellular response to stress through regulation of gene expression. Now, Huarte et al. (2010) identify a long intergenic noncoding RNA as a new player in p53-mediated repression of genes involved in apoptosis.

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Fyn-tau-amyloid: a toxic triad.

Publication Date: 2010 Aug 6 PMID: 20691893
Authors: Haass, C. - Mandelkow, E.
Journal: Cell

The axonal protein tau and amyloid beta-peptide (Abeta) are key players in the pathogenesis of Alzheimer's disease, and tau mediates Abeta toxicity, but it is not clear how. Ittner et al. (2010) now report an unexpected physiological function for tau in neuronal dendrites that may explain how tau mediates Abeta toxicity.

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Strategies for genetic studies of complex diseases.

Publication Date: 2010 Aug 6 PMID: 20691891
Authors: Wang, K. - Bucan, M. - Grant, S. F. - Schellenberg, G. - Hakonarson, H.
Journal: Cell

MeSH Categories: Disease/*genetics, *Genetic Heterogeneity, Humans

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Successes of genome-wide association studies.

Publication Date: 2010 Aug 6 PMID: 20691890
Authors: Klein, R. J. - Xu, X. - Mukherjee, S. - Willis, J. - Hayes, J.
Journal: Cell

MeSH Categories: Animals, *Genetic Predisposition to Disease, *Genome-Wide Association Study, Humans, Polymorphism, Single Nucleotide

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Stitching together cross-border research.

Publication Date: 2010 Aug 6 PMID: 20691889
Authors: Macilwain, C.
Journal: Cell

MeSH Categories: *Biomedical Research/economics/legislation & jurisprudence, Cooperative Behavior, European Union, Publications

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A shaker K+ channel with a miniature engineered voltage sensor.

Publication Date: 2010 Aug 20 PMID: 20691466
Authors: Xu, Y. - Ramu, Y. - Lu, Z.
Journal: Cell

Voltage-gated ion channels sense transmembrane voltage changes via a paddle-shaped motif that includes the C-terminal part of the third transmembrane segment (S3b) and the N-terminal part of the fourth segment ((NT)S4) that harbors voltage-sensing arginines. Here, we find that residue triplets in S3b and (NT)S4 can be deleted individually, or even in some combinations, without compromising the channels' basic voltage-gating capability. Thus, a high degree of complementarity between these S3b and (NT)S4 regions is not required for basic voltage gating per se. Remarkably, the voltage-gated Shaker K(+) channel remains voltage gated after a 43 residue paddle sequence is replaced by a glycine triplet. Therefore, the paddle motif comprises a minimal core that suffices to confer voltage gating in the physiological voltage range, and a larger, modulatory part. Our study also shows that the hydrophobic residues between the voltage-sensing arginines help set the sensor's characteristic chemical equilibrium between activated and deactivated states.

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A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response.

Publication Date: 2010 Aug 6 PMID: 20673990
Authors: Huarte, M. - Guttman, M. - Feldser, D. - Garber, M. - Koziol, M. J. - Kenzelmann-Broz, D. - Khalil, A. M. - Zuk, O. - Amit, I. - Rabani, M. - Attardi, L. D. - Regev, A. - Lander, E. S. - Jacks, T. - Rinn, J. L.
Journal: Cell

Recently, more than 1000 large intergenic noncoding RNAs (lincRNAs) have been reported. These RNAs are evolutionarily conserved in mammalian genomes and thus presumably function in diverse biological processes. Here, we report the identification of lincRNAs that are regulated by p53. One of these lincRNAs (lincRNA-p21) serves as a repressor in p53-dependent transcriptional responses. Inhibition of lincRNA-p21 affects the expression of hundreds of gene targets enriched for genes normally repressed by p53. The observed transcriptional repression by lincRNA-p21 is mediated through the physical association with hnRNP-K. This interaction is required for proper genomic localization of hnRNP-K at repressed genes and regulation of p53 mediates apoptosis. We propose a model whereby transcription factors activate lincRNAs that serve as key repressors by physically associating with repressive complexes and modulate their localization to sets of previously active genes.

MeSH Categories: Animals, Apoptosis, *Down-Regulation, Heterogeneous-Nuclear Ribonucleoprotein K/metabolism, Humans, Mice, Molecular Sequence Data, RNA, Untranslated/*metabolism, Transcription, Genetic, Tumor Suppressor Protein p53/*metabolism

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Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models.

Publication Date: 2010 Aug 6 PMID: 20655099
Authors: Ittner, L. M. - Ke, Y. D. - Delerue, F. - Bi, M. - Gladbach, A. - van Eersel, J. - Wolfing, H. - Chieng, B. C. - Christie, M. J. - Napier, I. A. - Eckert, A. - Staufenbiel, M. - Hardeman, E. - Gotz, J.
Journal: Cell

Alzheimer's disease (AD) is characterized by amyloid-beta (Abeta) and tau deposition in brain. It has emerged that Abeta toxicity is tau dependent, although mechanistically this link remains unclear. Here, we show that tau, known as axonal protein, has a dendritic function in postsynaptic targeting of the Src kinase Fyn, a substrate of which is the NMDA receptor (NR). Missorting of tau in transgenic mice expressing truncated tau (Deltatau) and absence of tau in tau(-/-) mice both disrupt postsynaptic targeting of Fyn. This uncouples NR-mediated excitotoxicity and hence mitigates Abeta toxicity. Deltatau expression and tau deficiency prevent memory deficits and improve survival in Abeta-forming APP23 mice, a model of AD. These deficits are also fully rescued with a peptide that uncouples the Fyn-mediated interaction of NR and PSD-95 in vivo. Our findings suggest that this dendritic role of tau confers Abeta toxicity at the postsynapse with direct implications for pathogenesis and treatment of AD.

MeSH Categories: Alzheimer Disease/pathology/*physiopathology, Amyloid beta-Protein/metabolism/toxicity, Animals, Brain/pathology, Dendrites/*metabolism, Humans, Intracellular Signaling Peptides and Proteins/metabolism, Membrane Proteins/metabolism, Memory Disorders/metabolism, Mice, Mice, Transgenic, Proto-Oncogene Proteins c-fyn/metabolism, Receptors, N-Methyl-D-Aspartate/metabolism, Synapses/metabolism, tau Proteins/genetics/*metabolism

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