ALBERT

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Changmeng Cai , Housheng Hansen He , Shuai Gao , Sen Chen , Ziyang Yu , Yanfei Gao , Shaoyong Chen , Mei Wei Chen , Jesse Zhang , Musaddeque Ahmed , Yang Wang , Eric Metzger , Roland Schüle , X. Shirley Liu , Myles Brown , Steven P. Balk Lysine-Specific Demethylase 1 (LSD1, KDM1A) functions as a transcriptional corepressor through demethylation of histone 3 lysine 4 (H3K4) but has a coactivator function on some genes through mechanisms that are unclear. We show that LSD1, interacting with CoREST, associates with and coactivates androgen receptor (AR) on a large fraction of androgen-stimulated genes. A subset of these AR/LSD1-associated enhancer sites have histone 3 threonine 6 phosphorylation (H3T6ph), and these sites are further enriched for androgen-stimulated genes. Significantly, despite its coactivator activity, LSD1 still mediates H3K4me2 demethylation at these androgen-stimulated enhancers. FOXA1 is also associated with LSD1 at AR-regulated enhancer sites, and a FOXA1 interaction with LSD1 enhances binding of both proteins at these sites. These findings show that LSD1 functions broadly as a regulator of AR function, that it maintains a transcriptional repression function at AR-regulated enhancers through H3K4 demethylation, and that it has a distinct AR-linked coactivator function mediated by demethylation of other substrates. Graphical abstract Teaser Cai et al. show that lysine-specific demethylase 1 (LSD1), although generally a transcriptional corepressor through H3K4 demethylation, functions broadly as a coactivator for androgen receptor and interacts with FOXA1 on androgen-stimulated genes. LSD1-mediated H3K4 demethylation persists at these sites, indicating a distinct coactivator function mediated by demethylation of other substrates.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Raajit Rampal , Altuna Alkalin , Jozef Madzo , Aparna Vasanthakumar , Elodie Pronier , Jay Patel , Yushan Li , Jihae Ahn , Omar Abdel-Wahab , Alan Shih , Chao Lu , Patrick S. Ward , Jennifer J. Tsai , Todd Hricik , Valeria Tosello , Jacob E. Tallman , Xinyang Zhao , Danette Daniels , Qing Dai , Luisa Ciminio , Iannis Aifantis , Chuan He , Francois Fuks , Martin S. Tallman , Adolfo Ferrando , Stephen Nimer , Elisabeth Paietta , Craig B. Thompson , Jonathan D. Licht , Christopher E. Mason , Lucy A. Godley , Ari Melnick , Maria E. Figueroa , Ross L. Levine Somatic mutations in IDH1/IDH2 and TET2 result in impaired TET2-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The observation that WT1 inactivating mutations anticorrelate with TET2/IDH1/IDH2 mutations in acute myeloid leukemia (AML) led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant AML patients have reduced 5hmC levels similar to TET2/IDH1/IDH2 mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations in DNA promoter methylation. WT1 overexpression increases global levels of 5hmC, and WT1 silencing reduced 5hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/IDH2 and WT1 mutations define an AML subtype defined by dysregulated DNA hydroxymethylation. Graphical abstract Teaser Mutational studies in patients with acute myeloid leukemia (AML) have identified recurrent mutations in TET2 and IDH1/IDH2 , and these mutations result in a reduction in 5-hydroxymethylcytosine (5hmC) levels. Rampal et al. demonstrate that WT1 mutations anticorrelate with TET2 and IDH1/IDH2 mutations, and WT1 mutant AMLs have decreased 5hmC levels, consistent with reduced TET2 function.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Shong Lau , Daniella Rylander Ottosson , Johan Jakobsson , Malin Parmar Recent findings show that human fibroblasts can be directly programmed into functional neurons without passing via a proliferative stem cell intermediate. These findings open up the possibility of generating subtype-specific neurons of human origin for therapeutic use from fetal cell, from patients themselves, or from matched donors. In this study, we present an improved system for direct neural conversion of human fibroblasts. The neural reprogramming genes are regulated by the neuron-specific microRNA, miR-124, such that each cell turns off expression of the reprogramming genes once the cell has reached a stable neuronal fate. The regulated system can be combined with integrase-deficient vectors, providing a nonintegrative and self-regulated conversion system that rids problems associated with the integration of viral transgenes into the host genome. These modifications make the system suitable for clinical use and therefore represent a major step forward in the development of induced neurons for cell therapy. Graphical abstract Teaser Lau et al. now use miRNA targeting to build a self-regulating neural conversion system. Combined with nonintegrating vectors, this system can efficiently drive conversion of human fibroblasts into functional induced neurons (iNs) suitable for clinical applications.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Lucas Leclère , Fabian Rentzsch Patterning of the metazoan dorsoventral axis is mediated by a complex interplay of BMP signaling regulators. Repulsive guidance molecule (RGM) is a conserved BMP coreceptor that has not been implicated in axis specification. We show that NvRGM is a key positive regulator of BMP signaling during secondary axis establishment in the cnidarian Nematostella vectensis . NvRGM regulates first the generation and later the shape of a BMP-dependent Smad1/5/8 gradient with peak activity on the side opposite the  NvBMP/NvRGM/NvChordin expression domain. Full knockdown of Smad1/5/8 signaling blocks the formation of endodermal structures, the mesenteries, and the establishment of bilateral symmetry, while altering the gradient through partial NvRGM or NvBMP knockdown shifts the boundaries of asymmetric gene expression and the positioning of the mesenteries along the secondary axis. These findings provide insight into the diversification of axis specification mechanisms and identify a previously unrecognized role for RGM in BMP-mediated axial patterning. Graphical abstract Teaser Leclère and Rentzsch identify repulsive guidance molecule (RGM) as an essential regulator of the BMP morphogen gradient that controls the formation and patterning of the secondary body axis in the sea anemone Nematostella . The evolutionary conservation of RGM-BMP interactions indicates that this function might also be important for bilaterian embryogenesis.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Grace E. Kim , Jack Kronengold , Giulia Barcia , Imran H. Quraishi , Hilary C. Martin , Edward Blair , Jenny C. Taylor , Olivier Dulac , Laurence Colleaux , Rima Nabbout , Leonard K. Kaczmarek Disease-causing mutations in ion channels generally alter intrinsic gating properties such as activation, inactivation, and voltage dependence. We examined nine different mutations of the KCNT1 (Slack) Na + -activated K + channel that give rise to three distinct forms of epilepsy. All produced many-fold increases in current amplitude compared to the wild-type channel. This could not be accounted for by increases in the intrinsic open probability of individual channels. Rather, greatly increased opening was a consequence of cooperative interactions between multiple channels in a patch. The degree of cooperative gating was much greater for all of the mutant channels than for the wild-type channel, and could explain increases in current even in a mutant with reduced unitary conductance. We also found that the same mutation gave rise to different forms of epilepsy in different individuals. Our findings indicate that a major consequence of these mutations is to alter channel-channel interactions. Graphical abstract Teaser Slack KCNT1 channels regulate how neurons respond to sustained stimulation. Kim et al. characterized nine KCNT1 mutations found in epilepsy patients with severe intellectual disabilities and showed that, in isolation, channel behavior is unaltered. However, in groups, mutant channels interact with each other abnormally, increasing current that flows through the channels.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Juanjuan Ou , Hongming Miao , Yinyan Ma , Feng Guo , Jia Deng , Xing Wei , Jie Zhou , Ganfeng Xie , Hang Shi , Bingzhong Xue , Houjie Liang , Liqing Yu How cancer cells shift metabolism to aerobic glycolysis is largely unknown. Here, we show that deficiency of α/β-hydrolase domain-containing 5 (Abhd5), an intracellular lipolytic activator that is also known as comparative gene identification 58 (CGI-58), promotes this metabolic shift and enhances malignancies of colorectal carcinomas (CRCs). Silencing of Abhd5 in normal fibroblasts induces malignant transformation. Intestine-specific knockout of Abhd5 in Apc Min/+ mice robustly increases tumorigenesis and malignant transformation of adenomatous polyps. In colon cancer cells, Abhd5 deficiency induces epithelial-mesenchymal transition by suppressing the AMPKα-p53 pathway, which is attributable to increased aerobic glycolysis. In human CRCs, Abhd5 expression falls substantially and correlates negatively with malignant features. Our findings link Abhd5 to CRC pathogenesis and suggest that cancer cells develop aerobic glycolysis by suppressing Abhd5-mediated intracellular lipolysis. Graphical abstract Teaser Cancer cells shift their metabolism to aerobic glycolysis (i.e., fermentation of glucose as energy in the presence of ample oxygen), but the underlying mechanisms remain elusive. Ou et al. identify Abhd5, an activator of intracellular fat breakdown, as a suppressor of this metabolic shift and associated malignancies in colon cancer.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Emmanuel Eggermann , Yves Kremer , Sylvain Crochet , Carl C.H. Petersen Internal brain states affect sensory perception, cognition, and learning. Many neocortical areas exhibit changes in the pattern and synchrony of neuronal activity during quiet versus active behaviors. Active behaviors are typically associated with desynchronized cortical dynamics. Increased thalamic firing contributes importantly to desynchronize mouse barrel cortex during active whisker sensing. However, a whisking-related cortical state change persists after thalamic inactivation, which is mediated at least in part by acetylcholine, as we show here by using whole-cell recordings, local pharmacology, axonal calcium imaging, and optogenetic stimulation. During whisking, we find prominent cholinergic signals in the barrel cortex, which suppress spontaneous cortical activity. The desynchronized state of barrel cortex during whisking is therefore driven by at least two distinct signals with opposing functions: increased thalamic activity driving glutamatergic excitation of the cortex and increased cholinergic input suppressing spontaneous cortical activity. Graphical abstract Teaser Eggermann et al. now find that the desynchronized state of the barrel cortex during active whisker sensing is accompanied by increased cholinergic input, which suppresses slow spontaneous cortical activity in excitatory layer 2/3 barrel cortex neurons.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Nancy A. Chow , Luke D. Jasenosky , Anne E. Goldfeld Interferon γ (IFN-γ) priming sensitizes monocytes and macrophages to lipopolysaccharide (LPS) stimulation, resulting in augmented expression of a set of genes including TNF . Here, we demonstrate that IFN-γ priming of LPS-stimulated TNF transcription requires a distal TNF/LT locus element 8 kb upstream of the TNF transcription start site (hHS-8). IFN-γ stimulation leads to increased DNase I accessibility of hHS-8 and its recruitment of interferon regulatory factor 1 (IRF1), and subsequent LPS stimulation enhances H3K27 acetylation and induces enhancer RNA synthesis at hHS-8. Ablation of IRF1 or targeting the hHS-8 IRF1 binding site in vivo with Cas9 linked to the KRAB repressive domain abolishes IFN-γ priming, but does not affect LPS induction of the gene. Thus, IFN-γ poises a distal enhancer in the TNF/LT locus by chromatin remodeling and IRF1 recruitment, which then drives enhanced TNF gene expression in response to a secondary toll-like receptor (TLR) stimulus. Graphical abstract Teaser Interferon γ (IFN-γ) priming is a critical immune event that enhances the monocyte and macrophage response, particularly expression of the TNF gene, to toll-like receptor (TLR) signaling. Chow et al. demonstrate that IFN-γ priming requires a distal enhancer element within the TNF/LT locus, thereby expanding the role of distal regulatory elements in the innate immune response.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Daniel W. Hagey , Jonas Muhr Organ formation and maintenance depends on slowly self-renewing stem cells that supply an intermediate population of rapidly dividing progenitors, but how this proliferative hierarchy is regulated is unknown. By performing genome-wide single-cell and functional analyses in the cortex, we demonstrate that reduced Sox2 expression is a key regulatory signature of the transition between stem cells and rapidly dividing progenitors. In stem cells, Sox2 is expressed at high levels, which enables its repression of proproliferative genes, of which Cyclin D1 is the most potent target. Sox2 confers this function through binding to low-affinity motifs, which facilitate the recruitment of Gro/Tle corepressors in synergy with Tcf/Lef proteins. Upon differentiation, proneural factors reduce Sox2 expression, which derepresses Cyclin D1 and promotes proliferation. Our results show how concentration-dependent Sox2 occupancy of DNA motifs of varying affinities translates into recruitment of repressive complexes, which regulate the proliferative dynamics of neural stem and progenitor cells. Graphical abstract Teaser Hagey and Muhr show that high levels of Sox2 maintain stem cells of the developing cortex in a slowly self-renewing state by directly repressing cell-cycle genes. They further demonstrate that proneural protein-induced commitment to differentiation induces a rapidly dividing state via the reduction of Sox2 expression levels.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Jakub O. Westholm , Pedro Miura , Sara Olson , Sol Shenker , Brian Joseph , Piero Sanfilippo , Susan E. Celniker , Brenton R. Graveley , Eric C. Lai Circularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues, and cultured cells, to rigorously annotate >2,500 fruit fly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and the circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1,000 well-conserved canonical miRNA seed matches, especially within coding regions, and coding conserved miRNA sites reside preferentially within circularized exons. Finally, we analyze the developmental and tissue specificity of circular RNAs and note their preferred derivation from neural genes and enhanced accumulation in neural tissues. Interestingly, circular isoforms increase substantially relative to linear isoforms during CNS aging and constitute an aging biomarker. Graphical abstract Teaser Westholm et al. annotate Drosophila circular RNAs from a massive collection of total RNA-seq data, providing insights into their biogenesis and function. In particular, circularizing exons are predominantly associated with long flanking introns, are preferred locations of conserved coding miRNA sites, and accumulate to highest levels in the aging CNS.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Bo-Kuan Wu , Charles Brenner Hypermethylation-mediated tumor suppressor gene (TSG) silencing is a central epigenetic alteration in RAS-dependent tumorigenesis. Ten-eleven translocation (TET) enzymes can depress DNA methylation by hydroxylation of 5-methylcytosine (5mC) bases to 5-hydroxymethylcytosine (5hmC). Here, we report that suppression of TET1 is required for KRAS-induced DNA hypermethylation and cellular transformation. In distinct nonmalignant cell lines, oncogenic KRAS promotes transformation by inhibiting TET1 expression via the ERK-signaling pathway. This reduces chromatin occupancy of TET1 at TSG promoters, lowers levels of 5hmC, and increases levels of 5mC and 5mC-dependent transcriptional silencing. Restoration of TET1 expression by ERK pathway inhibition or ectopic TET1 reintroduction in KRAS-transformed cells reactivates TSGs and inhibits colony formation. KRAS knockdown increases TET1 expression and diminishes colony-forming ability, whereas KRAS/TET1 double knockdown bypasses the KRAS dependence of KRAS-addicted cancer cells. Thus, suppression of TET1-dependent DNA demethylation is critical for KRAS-mediated transformation. Graphical abstract Teaser Wu and Brenner now show that KRAS drives ERK-dependent mRNA suppression of TET1 and that this is necessary to achieve tumor suppressor gene hypermethylation and malignant transformation.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Zekiye Buket Yılmaz , Bente Kofahl , Patrick Beaudette , Katharina Baum , Inbal Ipenberg , Falk Weih , Jana Wolf , Gunnar Dittmar , Claus Scheidereit The mechanisms that govern proteolytic maturation or complete destruction of the precursor proteins p100 and p105 are fundamental to homeostasis and activation of NF-κB; however, they remain poorly understood. Using mass-spectrometry-based quantitative analysis of noncanonical LTβR-induced signaling, we demonstrate that stimulation induces simultaneous processing of both p100 and p105. The precursors not only form hetero-oligomers but also interact with the ATPase VCP/p97, and their induced proteolysis strictly depends on the signal response domain (SRD) of p100, suggesting that the SRD-targeting proteolytic machinery acts in cis and in trans . Separation of cellular pools by isotope labeling revealed synchronous dynamics of p105 and p100 proteolysis. The generation of p50 and p52 from their precursors depends on functional VCP/p97. We have developed quantitative mathematical models that describe the dynamics of the system and predict that p100-p105 complexes are signal responsive. Graphical abstract Teaser Proteolytic precursor processing is a hallmark of the NF-κB system. Yilmaz et al. show that in lymphotoxin-stimulated cells p100 acts upstream of p105, resulting in concurrent production of p52 and p50. Both precursors form complexes and bind to segregase (p97/VCP), which promotes proteasomal processing. The findings are supported by mass spectrometry and incorporated in quantitative mathematical models.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Lisa L. Korn , Harper G. Hubbeling , Paige M. Porrett , Qi Yang , Lisa G. Barnett , Terri M. Laufer Regulatory T cells (Tregs) are CD4 + T cells that maintain immune homeostasis and prevent autoimmunity. Like all CD4 + T cells, Tregs require antigen-specific signals via T cell receptor-major histocompatibility complex class II (TCR-MHCII) interactions for their development. However, the requirement for MHCII in Treg homeostasis in tissues such as intestinal lamina propria (LP) is unknown. We examined LP Treg homeostasis in a transgenic mouse model that lacks peripheral TCR-MHCII interactions and generation of extrathymic Tregs (iTregs). Thymically generated Tregs entered the LP of weanlings and proliferated independently of MHCII to fill the compartment. The adult LP was a closed niche; new thymic Tregs were excluded, and Tregs in parabiotic pairs were LP resident. The isolated LP niche was interleukin-2 (IL-2) independent but dependent on commensal bacteria. Thus, an LP Treg niche can be filled, isolated, and maintained independently of antigen signals and iTregs. This niche may represent a tissue-specific mechanism for maintaining immune tolerance. Graphical abstract Teaser Regulatory T cells (Tregs) maintain immune homeostasis and prevent autoimmunity. Korn et al. describe a unique Treg niche in the intestinal lamina propria that does not require T cell receptor signals for development or maintenance and is physiologically isolated from the Tregs that circulate through lymphoid organs. Maintenance of this niche is dependent upon local commensal bacteria. The authors propose that this isolated niche may represent a tissue-specific mechanism for maintaining immune tolerance.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Romain Christiano , Nagarjuna Nagaraj , Florian Fröhlich , Tobias C. Walther How cells maintain specific levels of each protein and whether that control is evolutionarily conserved are key questions. Here, we report proteome-wide steady-state protein turnover rate measurements for the evolutionarily distant but ecologically similar yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe . We find that the half-life of most proteins is much longer than currently thought and determined to a large degree by protein synthesis and dilution due to cell division. However, we detect a significant subset of proteins (∼15%) in both yeasts that are turned over rapidly. In addition, the relative abundances of orthologous proteins between the two yeasts are highly conserved across the 400 million years of evolution. In contrast, their respective turnover rates differ considerably. Our data provide a high-confidence resource for studying protein degradation in common yeast model systems. Graphical abstract Teaser Christiano et al. report protein turnover rates for S. cerevisiae and S. pombe . Overall, protein half-life is far less conserved than protein abundance. Protein turnover profiling of hrd1 Δ, an endoplasmic-reticulum-associated degradation (ERAD)-defective mutant, identifies candidate substrates for this pathway in budding yeast.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Brian C. Shonesy , Rebecca J. Bluett , Teniel S. Ramikie , Rita Báldi , Daniel J. Hermanson , Philip J. Kingsley , Lawrence J. Marnett , Danny G. Winder , Roger J. Colbran , Sachin Patel Endocannabinoid (eCB) signaling has been heavily implicated in the modulation of anxiety and depressive behaviors and emotional learning. However, the role of the most-abundant endocannabinoid 2-arachidonoylglycerol (2-AG) in the physiological regulation of affective behaviors is not well understood. Here, we show that genetic deletion of the 2-AG synthetic enzyme diacylglycerol lipase α (DAGLα) in mice reduces brain, but not circulating, 2-AG levels. DAGLα deletion also results in anxiety-like and sex-specific anhedonic phenotypes associated with impaired activity-dependent eCB retrograde signaling at amygdala glutamatergic synapses. Importantly, acute pharmacological normalization of 2-AG levels reverses both phenotypes of DAGLα-deficient mice. These data suggest 2-AG deficiency could contribute to the pathogenesis of affective disorders and that pharmacological normalization of 2-AG signaling could represent an approach for the treatment of mood and anxiety disorders. Graphical abstract Teaser The role of the primary endogenous cannabinoid 2-AG in mood and anxiety regulation is not well understood. Shonesy et al. show that deletion of a primary 2-AG synthetic enzyme, DAGLα, results in anxiety and sex-specific depressive phenotypes, which can be rapidly reversed by pharmacological normalization of endocannabinoid levels.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Kiran Batta , Magdalena Florkowska , Valerie Kouskoff , Georges Lacaud Recent reports have shown that somatic cells, under appropriate culture conditions, could be directly reprogrammed to cardiac, hepatic, or neuronal phenotype by lineage-specific transcription factors. In this study, we demonstrate that both embryonic and adult somatic fibroblasts can be efficiently reprogrammed to clonal multilineage hematopoietic progenitors by the ectopic expression of the transcription factors ERG, GATA2, LMO2, RUNX1c, and SCL. These reprogrammed cells were stably expanded on stromal cells and possessed short-term reconstitution ability in vivo. Loss of p53 function facilitated reprogramming to blood, and p53 −/− reprogrammed cells efficiently generated erythroid, megakaryocytic, myeloid, and lymphoid lineages. Genome-wide analyses revealed that generation of hematopoietic progenitors was preceded by the appearance of hemogenic endothelial cells expressing endothelial and hematopoietic genes. Altogether, our findings suggest that direct reprogramming could represent a valid alternative approach to the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for disease modeling and autologous blood cell therapies. Graphical abstract Teaser Batta et al. demonstrate that murine fibroblasts are reprogrammed to hematopoietic progenitors, with erythroid, megakaryocyte, and myeloid potential, by ectopic expression of hematopoietic transcription factors. Reprogramming efficiency is increased by loss of p53 function, and generation of blood cells is preceded by the appearance of hemogenic endothelium.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Nancy F. Ramia , Michael Spilman , Li Tang , Yaming Shao , Joshua Elmore , Caryn Hale , Alexis Cocozaki , Nilakshee Bhattacharya , Rebecca M. Terns , Michael P. Terns , Hong Li , Scott M. Stagg The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA) and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes. Graphical abstract Teaser Ramia et al. show that the helical core of the type III-B Cmr CRISPR-Cas effector complex, made up of multiple Cmr4 subunits, forms the platform for a corresponding number of cleavages of the target RNA. Comparison with the type I-E Cascade structure reveals strikingly similar mechanisms of crRNA and target binding.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Christos G. Gkogkas , Arkady Khoutorsky , Ruifeng Cao , Seyed Mehdi Jafarnejad , Masha Prager-Khoutorsky , Nikolaos Giannakas , Archontia Kaminari , Apostolia Fragkouli , Karim Nader , Theodore J. Price , Bruce W. Konicek , Jeremy R. Graff , Athina K. Tzinia , Jean-Claude Lacaille , Nahum Sonenberg Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS phenotypes. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1 −/y ), we show that phosphorylation of the mRNA 5′ cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1 −/y mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS. Graphical abstract Teaser Fragile X syndrome (FXS) is caused by dysregulation of translation in the brain. Gkogkas et al. show that phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) is increased in FXS postmortem brains and Fmr1 −/y mice. Downregulation of eIF4E phosphorylation in Fmr1 −/y mice rescues defects in dendritic spine morphology, synaptic plasticity, and social interaction via normalization of MMP-9 expression.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Wei-Qun Fang , Wei-Wei Chen , Liwen Jiang , Kai Liu , Wing-Ho Yung , Amy K.Y. Fu , Nancy Y. Ip The functional integrity of the neocortex depends upon proper numbers of excitatory and inhibitory neurons; however, the consequences of dysregulated neuronal production during the development of the neocortex are unclear. As excess cortical neurons are linked to the neurodevelopmental disorder autism, we investigated whether the overproduction of neurons leads to neocortical malformation and malfunction in mice. We experimentally increased the number of pyramidal neurons in the upper neocortical layers by using the small molecule XAV939 to expand the intermediate progenitor population. The resultant overpopulation of neurons perturbs development of dendrites and spines of excitatory neurons and alters the laminar distribution of interneurons. Furthermore, these phenotypic changes are accompanied by dysregulated excitatory and inhibitory synaptic connection and balance. Importantly, these mice exhibit behavioral abnormalities resembling those of human autism. Thus, our findings collectively suggest a causal relationship between neuronal overproduction and autism-like features, providing developmental insights into the etiology of autism. Graphical abstract Teaser Fang et al. generated a mouse model with excessive excitatory neurons in the neocortex by manipulating embryonic neurogenesis. Overproduction of neurons results in autism-like anatomical and behavioral features. These findings suggest a causal relationship between overproduction of neurons and cortical malfunction and provide developmental insights into the etiology of autism.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Lauren J. Manderfield , Kurt A. Engleka , Haig Aghajanian , Mudit Gupta , Steven Yang , Li Li , Julie E. Baggs , John B. Hogenesch , Eric N. Olson , Jonathan A. Epstein Loss of Pax3, a developmentally regulated transcription factor expressed in premigratory neural crest, results in severe developmental defects and embryonic lethality. Although Pax3 mutations produce profound phenotypes, the intrinsic transcriptional activation exhibited by Pax3 is surprisingly modest. We postulated the existence of transcriptional coactivators that function with Pax3 to mediate developmental functions. A high-throughput screen identified the Hippo effector proteins Taz and Yap65 as Pax3 coactivators. Synergistic coactivation of target genes by Pax3-Taz/Yap65 requires DNA binding by Pax3, is Tead independent, and is regulated by Hippo kinases Mst1 and Lats2. In vivo, Pax3 and Yap65 colocalize in the nucleus of neural crest progenitors in the dorsal neural tube. Neural crest deletion of Taz and Yap65 results in embryo-lethal neural crest defects and decreased expression of the Pax3 target gene, Mitf . These results suggest that Pax3 activity is regulated by the Hippo pathway and that Pax factors are Hippo effectors. Graphical abstract Teaser Hippo signaling is a conserved kinase cascade that mediates transcription through a Yap65/Taz-Tead complex and governs organ size. Manderfield et al. have identified Yap65 and Taz as coactivators of Pax factors and established neural crest as a novel site of Hippo signaling. The Pax3-Yap65/Taz complex is regulated by upstream Hippo kinases and is Tead independent. Deletion of Yap65 and Taz in neural crest generated differentiation defects in specific neural crest derivatives.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Chris M. Woodard , Brian A. Campos , Sheng-Han Kuo , Melissa J. Nirenberg , Michael W. Nestor , Matthew Zimmer , Eugene V. Mosharov , David Sulzer , Hongyan Zhou , Daniel Paull , Lorraine Clark , Eric E. Schadt , Sergio Pablo Sardi , Lee Rubin , Kevin Eggan , Mathew Brock , Scott Lipnick , Mahendra Rao , Stephen Chang , Aiqun Li , Scott A. Noggle Parkinson’s disease (PD) has been attributed to a combination of genetic and nongenetic factors. We studied a set of monozygotic twins harboring the heterozygous glucocerebrosidase mutation ( GBA N370S) but clinically discordant for PD. We applied induced pluripotent stem cell (iPSC) technology for PD disease modeling using the twins’ fibroblasts to evaluate and dissect the genetic and nongenetic contributions. Utilizing fluorescence-activated cell sorting, we obtained a homogenous population of “footprint-free” iPSC-derived midbrain dopaminergic (mDA) neurons. The mDA neurons from both twins had ∼50% GBA enzymatic activity, ∼3-fold elevated α-synuclein protein levels, and a reduced capacity to synthesize and release dopamine. Interestingly, the affected twin’s neurons showed an even lower dopamine level, increased monoamine oxidase B (MAO-B) expression, and impaired intrinsic network activity. Overexpression of wild-type GBA and treatment with MAO-B inhibitors normalized α-synuclein and dopamine levels, suggesting a combination therapy for the affected twin. Graphical abstract Teaser Woodard et al. studied a set of identical twins harboring GBA N370S mutation but clinically discordant for Parkinson's disease (PD). Using iPSC technology, they found that enzymes GBA and MAO-B could be therapeutic targets in PD. These findings shed light into future studies using iPSCs to model idiopathic PD cases.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Hyokjoon Kwon , Sarnia Laurent , Yan Tang , Haihong Zong , Pratibha Vemulapalli , Jeffrey E. Pessin Adipose tissue inflammation is one pathway shown to mediate insulin resistance in obese humans and rodents. Obesity induces dynamic cellular changes in adipose tissue to increase proinflammatory cytokines and diminish anti-inflammatory cytokines. However, we have found that anti-inflammatory interleukin-13 (IL-13) is unexpectedly induced in adipose tissue of obese humans and high-fat diet (HFD)-fed mice, and the source of IL-13 is primarily the adipocyte. Moreover, HFD-induced proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and IL-1β mediate IL-13 production in adipocytes in an IKKβ-dependent manner. In contrast, adipocyte-specific IKKβ-deficient mice show diminished IL-13 expression and enhanced inflammation after HFD feeding, resulting in a worsening of the insulin-resistant state. Together these data demonstrate that although IKKβ activates the expression of proinflammatory mediators, in adipocytes, IKKβ signaling also induces the expression of the anti-inflammatory cytokine IL-13, which plays a unique protective role by limiting adipose tissue inflammation and insulin resistance. Graphical abstract Teaser IKKβ is known to be a proinflammatory mediator. However, IKKβ in adipocytes also mediates IL-13 expression to suppress high-fat-diet-induced inflammation in adipose tissue. This feedback mechanism may be the molecular basis of diet-induced chronic low-grade inflammation, resulting in systemic insulin resistance.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Morgane Belle , David Godefroy , Chloé Dominici , Céline Heitz-Marchaland , Pavol Zelina , Farida Hellal , Frank Bradke , Alain Chédotal Clearing techniques have been developed to transparentize mouse brains, thereby preserving 3D structure, but their complexity has limited their use. Here, we show that immunolabeling of axonal tracts followed by optical clearing with solvents (3DISCO) and light-sheet microscopy reveals brain connectivity in mouse embryos and postnatal brains. We show that the Robo3 receptor is selectively expressed by medial habenula axons forming the fasciculus retroflexus (FR) and analyzed the development of this commissural tract in mutants of the Slit/Robo and DCC/Netrin pathways. Netrin-1 and DCC are required to attract FR axons to the midline, but the two mutants exhibit specific and heterogeneous axon guidance defects. Moreover, floor-plate-specific deletion of Slit ligands with a conditional Slit2 allele perturbs not only midline crossing by FR axons but also their anteroposterior distribution. In conclusion, this method represents a unique and powerful imaging tool to study axonal connectivity in mutant mice. Graphical abstract Teaser Clearing techniques have recently been developed to look at mouse brains, but they are complex and expensive. Belle et al. now describe a simple procedure that combines immunolabeling, solvent-based clearing, and light-sheet fluorescence microscopy. This technique allows large-scale screening of axon guidance defects and other developmental disorders in mutant mice.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Emmanuelle Godefroy , Anne Gallois , Juliana Idoyaga , Miriam Merad , Navpreet Tung , Ngozi Monu , Yvonne Saenger , Yichun Fu , Rajesh Ravindran , Bali Pulendran , Francine Jotereau , Sergio Trombetta , Nina Bhardwaj Matrix metalloproteinase-2 (MMP-2) is involved in several physiological mechanisms, including wound healing and tumor progression. We show that MMP-2 directly stimulates dendritic cells (DCs) to both upregulate OX40L on the cell surface and secrete inflammatory cytokines. The mechanism underlying DC activation includes physical association with Toll-like receptor-2 (TLR2), leading to NF-κB activation, OX40L upregulation on DCs, and ensuing T H 2 differentiation. Significantly, MMP-2 polarizes T cells toward type 2 responses in vivo, in a TLR2-dependent manner. MMP-2-dependent type 2 polarization may represent a key immune regulatory mechanism for protection against a broad array of disorders, such as inflammatory, infectious, and autoimmune diseases, which can be hijacked by tumors to evade immunity. Graphical abstract Teaser Godefroy et al. now demonstrate that matrix metalloproteinase-2 (MMP-2) directly interacts with and activates dendritic cells (DCs) via Toll-like receptor-2. MMP-2-exposed DCs upregulate OX40L, promoting type 2 polarization both in vitro and in vivo. This may represent a key immune regulatory mechanism involved in a variety of inflammatory disorders.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Catalin Chimerel , Edward Emery , David K. Summers , Ulrich Keyser , Fiona M. Gribble , Frank Reimann It has long been speculated that metabolites, produced by gut microbiota, influence host metabolism in health and diseases. Here, we reveal that indole, a metabolite produced from the dissimilation of tryptophan, is able to modulate the secretion of glucagon-like peptide-1 (GLP-1) from immortalized and primary mouse colonic L cells. Indole increased GLP-1 release during short exposures, but it reduced secretion over longer periods. These effects were attributed to the ability of indole to affect two key molecular mechanisms in L cells. On the one hand, indole inhibited voltage-gated K + channels, increased the temporal width of action potentials fired by L cells, and led to enhanced Ca 2+ entry, thereby acutely stimulating GLP-1 secretion. On the other hand, indole slowed ATP production by blocking NADH dehydrogenase, thus leading to a prolonged reduction of GLP-1 secretion. Our results identify indole as a signaling molecule by which gut microbiota communicate with L cells and influence host metabolism. Graphical abstract Teaser Indole is the main metabolite produced by gut bacteria from tryptophan. Chimerel et al. demonstrate that indole modulates the hormone secretion of enteroendocrine L cells and reveal the molecular mechanism behind this modulation. These findings suggest that the production of indole by bacteria could have a major impact on host metabolism.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Qing-ming Gao , Keshun Yu , Ye Xia , M.B. Shine , Caixia Wang , DuRoy Navarre , Aardra Kachroo , Pradeep Kachroo The plant galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) have been linked to the anti-inflammatory and cancer benefits of a green leafy vegetable diet in humans due to their ability to regulate the levels of free radicals like nitric oxide (NO). Here, we show that DGDG contributes to plant NO as well as salicylic acid biosynthesis and is required for the induction of systemic acquired resistance (SAR). In contrast, MGDG regulates the biosynthesis of the SAR signals azelaic acid (AzA) and glycerol-3-phosphate (G3P) that function downstream of NO. Interestingly, DGDG is also required for AzA-induced SAR, but MGDG is not. Notably, transgenic expression of a bacterial glucosyltransferase is unable to restore SAR in dgd1 plants even though it does rescue their morphological and fatty acid phenotypes. These results suggest that MGDG and DGDG are required at distinct steps and function exclusively in their individual roles during the induction of SAR. Graphical abstract Teaser The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) constitute ∼80% of total membrane lipids in plants. Gao et al. now show that these galactolipids function nonredundantly to regulate systemic acquired resistance (SAR). Furthermore, they show that the terminal galactose on the α-galactose-β-galactose head group of DGDG is critical for SAR.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Rafael B. Blasco , Elif Karaca , Chiara Ambrogio , Taek-Chin Cheong , Emre Karayol , Valerio G. Minero , Claudia Voena , Roberto Chiarle Generation of genetically engineered mouse models (GEMMs) for chromosomal translocations in the endogenous loci by a knockin strategy is lengthy and costly. The CRISPR/Cas9 system provides an innovative and flexible approach for genome engineering of genomic loci in vitro and in vivo. Here, we report the use of the CRISPR/Cas9 system for engineering a specific chromosomal translocation in adult mice in vivo. We designed CRISPR/Cas9 lentiviral vectors to induce cleavage of the murine endogenous Eml4 and Alk loci in order to generate the Eml4-Alk gene rearrangement recurrently found in non-small-cell lung cancers (NSCLCs). Intratracheal or intrapulmonary inoculation of lentiviruses induced Eml4-Alk gene rearrangement in lung cells in vivo. Genomic and mRNA sequencing confirmed the genome editing and the production of the Eml4-Alk fusion transcript. All mice developed Eml4-Alk -rearranged lung tumors 2 months after the inoculation, demonstrating that the CRISPR/Cas9 system is a feasible and simple method for the generation of chromosomal rearrangements in vivo. Graphical abstract Teaser Blasco et al. demonstrate that CRISPR/Cas9 technology can be exploited to generate targeted chromosomal rearrangements in vivo in mice in a time- and cost-effective approach.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Andrea Galmozzi , Si B. Sonne , Svetlana Altshuler-Keylin , Yutaka Hasegawa , Kosaku Shinoda , Ineke H.N. Luijten , Jae Won Chang , Louis Z. Sharp , Benjamin F. Cravatt , Enrique Saez , Shingo Kajimura Obesity develops when energy intake chronically exceeds energy expenditure. Because brown adipose tissue (BAT) dissipates energy in the form of heat, increasing energy expenditure by augmenting BAT-mediated thermogenesis may represent an approach to counter obesity and its complications. The ability of BAT to dissipate energy is dependent on expression of mitochondrial uncoupling protein 1 (UCP1). To facilitate the identification of pharmacological modulators of BAT UCP1 levels, which may have potential as antiobesity medications, we developed a transgenic model in which luciferase activity faithfully mimics endogenous UCP1 expression and its response to physiologic stimuli. Phenotypic screening of a library using cells derived from this model yielded a small molecule that increases UCP1 expression in brown fat cells and mice. Upon adrenergic stimulation, compound-treated mice showed increased energy expenditure. These tools offer an opportunity to identify pharmacologic modulators of UCP1 expression and uncover regulatory pathways that impact BAT-mediated thermogenesis. Graphical abstract Teaser Pharmacological activation of brown adipose tissue (BAT) thermogenesis and energy dissipation, a process mediated by UCP1, may be useful to counter the energy imbalance that engenders obesity. Galmozzi et al. have developed an in vivo model to monitor UCP1 expression in real time and identified a small molecule that increases UCP1 levels. Mice treated with this molecule show greater energy expenditure upon adrenergic stimulation. Discovery of compounds with this ability is an important stride toward enhancing BAT function in obese individuals.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Yanmeng Guo , Yuping Wang , Qingxiu Wang , Zuoren Wang In Drosophila larvae, the class IV dendritic arborization (da) neurons are polymodal nociceptors. Here, we show that ppk26 (CG8546) plays an important role in mechanical nociception in class IV da neurons. Our immunohistochemical and functional results demonstrate that ppk26 is specifically expressed in class IV da neurons. Larvae with mutant ppk26 showed severe behavioral defects in a mechanical nociception behavioral test but responded to noxious heat stimuli comparably to wild-type larvae. In addition, functional studies suggest that ppk26 and ppk (also called ppk1 ) function in the same pathway, whereas piezo functions in a parallel pathway. Consistent with these functional results, we found that PPK and PPK26 are interdependent on each other for their cell surface localization. Our work indicates that PPK26 and PPK might form heteromeric DEG/ENaC channels that are essential for mechanotransduction in class IV da neurons. Graphical abstract Teaser Sensing painful stimuli is of vital importance for animal survival. Guo et al. now find that PPK26 is selectively expressed in class IV dendritic arborization neurons and contributes to mechanical nociception but not thermal nociception in Drosophila larvae.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Amol Ugale , Gudmundur L. Norddahl , Martin Wahlestedt , Petter Säwén , Pekka Jaako , Cornelis Jan Pronk , Shamit Soneji , Jörg Cammenga , David Bryder Studies of developmental pathways of hematopoietic stem cells (HSCs) have defined lineage relationships throughout the blood system. This is relevant to acute myeloid leukemia (AML), where aggressiveness and therapeutic responsiveness can be influenced by the initial stage of transformation. To address this, we generated a mouse model in which the mixed-lineage leukemia/eleven-nineteen-leukemia (MLL-ENL) transcription factor can be conditionally activated in any cell type. We show that AML can originate from multiple hematopoietic progenitor subsets with granulocytic and monocytic potential, and that the normal developmental position of leukemia-initiating cells influences leukemic development. However, disease failed to arise from HSCs. Although it maintained or upregulated the expression of target genes associated with leukemic development, MLL-ENL dysregulated the proliferative and repopulating capacity of HSCs. Therefore, the permissiveness for development of AML may be associated with a narrower window of differentiation than was previously appreciated, and hijacking the self-renewal capacity of HSCs by a potent oncogene is insufficient for leukemic development. Graphical abstract Teaser The cellular origin of leukemia driven by MLL fusions has been suggested to underlie heterogeneity in aggressiveness and prognosis. Ugale et al. now use an inducible MLL-ENL mouse model to study leukemia initiation and competence throughout the hematopoietic hierarchy. Although AML development could originate from multiple progenitor subsets, the most primitive stem cells were unexpectedly unable to initiate disease.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Nila Roy Choudhury , Jakub S. Nowak , Juan Zuo , Juri Rappsilber , Steven H. Spoel , Gracjan Michlewski RNA binding proteins have thousands of cellular RNA targets and often exhibit opposite or passive molecular functions. Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in cis , thereby providing an additional level of specificity. Graphical abstract Teaser Lin28a triggers TuT4-mediated pre-let-7 uridylation. Despite binding to other pre-microRNAs, only pre-let-7 is efficiently uridylated by TuT4. Choudhury et al. show that Trim25 is an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. These findings reveal that Trim25 can guide the function of canonical RNP complexes in cis , thereby providing an additional level of specificity.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Anita Parmigiani , Aida Nourbakhsh , Boxiao Ding , Wei Wang , Young Chul Kim , Konstantin Akopiants , Kun-Liang Guan , Michael Karin , Andrei V. Budanov The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a sensor of different environmental conditions and regulator of cell growth, metabolism, and autophagy. mTORC1 is activated by Rag GTPases, working as RagA:RagB and RagC:RagD heterodimers. Rags control mTORC1 activity by tethering mTORC1 to the lysosomes where it is activated by Rheb GTPase. RagA:RagB, active in its GTP-bound form, is inhibited by GATOR1 complex, a GTPase-activating protein, and GATOR1 is in turn negatively regulated by GATOR2 complex. Sestrins are stress-responsive proteins that inhibit mTORC1 via activation of AMP-activated protein kinase (AMPK) and tuberous sclerosis complex. Here we report an AMPK-independent mechanism of mTORC1 inhibition by Sestrins mediated by their interaction with GATOR2. As a result of this interaction, the Sestrins suppress mTOR lysosomal localization in a Rag-dependent manner. This mechanism is potentially involved in mTORC1 regulation by amino acids, rotenone, and tunicamycin, connecting stress response with mTORC1 inhibition. Graphical abstract Teaser The mTORC1 kinase integrates various environmental signals to regulate cell growth and metabolism. Parmigiani et al. identified a mechanism of mTORC1 regulation by Sestrins via interaction with GATOR2 and suppression of mTOR lysosomal localization.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Nicholas J. Bessman , Atrish Bagchi , Kathryn M. Ferguson , Mark A. Lemmon The epidermal growth factor receptor (EGFR) plays pivotal roles in development and is mutated or overexpressed in several cancers. Despite recent advances, the complex allosteric regulation of EGFR remains incompletely understood. Through efforts to understand why the negative cooperativity observed for intact EGFR is lost in studies of its isolated extracellular region (ECR), we uncovered unexpected relationships between ligand binding and receptor dimerization. The two processes appear to compete. Surprisingly, dimerization does not enhance ligand binding (although ligand binding promotes dimerization). We further show that simply forcing EGFR ECRs into preformed dimers without ligand yields ill-defined, heterogeneous structures. Finally, we demonstrate that extracellular EGFR-activating mutations in glioblastoma enhance ligand-binding affinity without directly promoting EGFR dimerization, suggesting that these oncogenic mutations alter the allosteric linkage between dimerization and ligand binding. Our findings have important implications for understanding how EGFR and its relatives are activated by specific ligands and pathological mutations. Graphical abstract Teaser Although the epidermal growth factor receptor (EGFR) was the first growth factor receptor for which ligand-induced dimerization was established, the relationship between growth factor binding and EGFR dimerization remains unclear. Bessman et al. use a range of biophysical methods to study growth factor binding to EGFR variants, including those found in glioblastoma, to shed light on ligand-specific allosteric control of EGFR.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Kristen L. Karlin , Gourish Mondal , Jessica K. Hartman , Siddhartha Tyagi , Sarah J. Kurley , Chris S. Bland , Tiffany Y.T. Hsu , Alexander Renwick , Justin E. Fang , Ilenia Migliaccio , Celetta Callaway , Amritha Nair , Rocio Dominguez-Vidana , Don X. Nguyen , C. Kent Osborne , Rachel Schiff , Li-Yuan Yu-Lee , Sung Y. Jung , Dean P. Edwards , Susan G. Hilsenbeck , Jeffrey M. Rosen , Xiang H.-F. Zhang , Chad A. Shaw , Fergus J. Couch , Thomas F. Westbrook Defining the molecular networks that drive breast cancer has led to therapeutic interventions and improved patient survival. However, the aggressive triple-negative breast cancer subtype (TNBC) remains recalcitrant to targeted therapies because its molecular etiology is poorly defined. In this study, we used a forward genetic screen to discover an oncogenic network driving human TNBC. S CYL1, T EX14, and P LK1 (“STP axis”) cooperatively trigger degradation of the REST tumor suppressor protein, a frequent event in human TNBC. The STP axis induces REST degradation by phosphorylating a conserved REST phospho-degron and bridging REST interaction with the ubiquitin-ligase βTRCP. Inhibition of the STP axis leads to increased REST protein levels and impairs TNBC transformation, tumor progression, and metastasis. Expression of the STP axis correlates with low REST protein levels in human TNBCs and poor clinical outcome for TNBC patients. Our findings demonstrate that the STP-REST axis is a molecular driver of human TNBC. Graphical abstract Teaser Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype for which the molecular drivers are poorly understood. Karlin et al. now demonstrate that S CYL1, T EX14, and P LK1 (“STP axis”) cooperatively trigger degradation of the REST tumor suppressor protein, a frequent event that may provide a therapeutic entry point for human TNBC.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Christina L. Zheng , Nicholas J. Wang , Jongsuk Chung , Homayoun Moslehi , J. Zachary Sanborn , Joseph S. Hur , Eric A. Collisson , Swapna S. Vemula , Agne Naujokas , Kami E. Chiotti , Jeffrey B. Cheng , Hiva Fassihi , Andrew J. Blumberg , Celeste V. Bailey , Gary M. Fudem , Frederick G. Mihm , Bari B. Cunningham , Isaac M. Neuhaus , Wilson Liao , Dennis H. Oh , James E. Cleaver , Philip E. LeBoit , Joseph F. Costello , Alan R. Lehmann , Joe W. Gray , Paul T. Spellman , Sarah T. Arron , Nam Huh , Elizabeth Purdom , Raymond J. Cho Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC −/− background. XPC −/− cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk. Graphical abstract Teaser Zheng et al. report that variable mutation densities within cancer genomes result from differential access of DNA repair machinery, imposed by chromatin state. By showing that transcription restores DNA repair to tightly packaged DNA, their study reveals natural differences in expression level as a potentially important modulator of oncogene mutation rate.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Ana Freije , Rut Molinuevo , Laura Ceballos , Marta Cagigas , Pilar Alonso-Lecue , René Rodriguez , Pablo Menendez , Daniel Aberdam , Ernesto De Diego , Alberto Gandarillas Tumor suppressor p53 is a major cellular guardian of genome integrity, and its inactivation is the most frequent genetic alteration in cancer, rising up to 80% in squamous cell carcinoma (SCC). By adapting the small hairpin RNA (shRNA) technology, we inactivated endogenous p53 in primary epithelial cells from the epidermis of human skin. We show that either loss of endogenous p53 or overexpression of a temperature-sensitive dominant-negative conformation triggers a self-protective differentiation response, resulting in cell stratification and expulsion. These effects follow DNA damage and exit from mitosis without cell division. p53 preserves the proliferative potential of the stem cell compartment and limits the power of proto-oncogene MYC to drive cell cycle stress and differentiation. The results provide insight into the role of p53 in self-renewal homeostasis and help explain why p53 mutations do not initiate skin cancer but increase the likelihood that cancer cells will appear. Graphical abstract Teaser The p53 tumor suppressor is frequently inactivated in squamous cell carcinoma, yet loss of p53 does not initiate nonmelanoma skin cancer, suggesting that epithelial skin cells have self-protective mechanisms. Freije et al. show that p53 enhances proliferation and inhibits differentiation in keratinocytes by preventing endoreplication. p53 loss leads to squamous differentiation and expulsion of mutant cells, which may confer the epidermis with a molecular protective response.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Andreas Gewies , Oliver Gorka , Hanna Bergmann , Konstanze Pechloff , Franziska Petermann , Katharina M. Jeltsch , Martina Rudelius , Mark Kriegsmann , Wilko Weichert , Marion Horsch , Johannes Beckers , Wolfgang Wurst , Mathias Heikenwalder , Thomas Korn , Vigo Heissmeyer , Jürgen Ruland The paracaspase Malt1 is a central regulator of antigen receptor signaling that is frequently mutated in human lymphoma. As a scaffold, it assembles protein complexes for NF-κB activation, and its proteolytic domain cleaves negative NF-κB regulators for signal enforcement. Still, the physiological functions of Malt1-protease are unknown. We demonstrate that targeted Malt1-paracaspase inactivation induces a lethal inflammatory syndrome with lymphocyte-dependent neurodegeneration in vivo. Paracaspase activity is essential for regulatory T cell (Treg) and innate-like B cell development, but it is largely dispensable for overcoming Malt1-dependent thresholds for lymphocyte activation. In addition to NF-κB inhibitors, Malt1 cleaves an entire set of mRNA stability regulators, including Roquin-1, Roquin-2, and Regnase-1, and paracaspase inactivation results in excessive interferon gamma (IFNγ) production by effector lymphocytes that drive pathology. Together, our results reveal distinct threshold and modulatory functions of Malt1 that differentially control lymphocyte differentiation and activation pathways and demonstrate that selective paracaspase blockage skews systemic immunity toward destructive autoinflammation. Graphical abstract Teaser The paracaspase Malt1 is a key regulator of antigen receptor signaling and frequently mutated in human lymphoma. Gewies et al. demonstrate that the proteolytic function of Malt1 is largely dispensable for lymphocyte activation but critical for regulatory T cell (Treg) and innate-like B cell development and protection from IFNγ-mediated autoinflammation in vivo.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Anne Biton , Isabelle Bernard-Pierrot , Yinjun Lou , Clémentine Krucker , Elodie Chapeaublanc , Carlota Rubio-Pérez , Nuria López-Bigas , Aurélie Kamoun , Yann Neuzillet , Pierre Gestraud , Luca Grieco , Sandra Rebouissou , Aurélien de Reyniès , Simone Benhamou , Thierry Lebret , Jennifer Southgate , Emmanuel Barillot , Yves Allory , Andrei Zinovyev , François Radvanyi Extracting relevant information from large-scale data offers unprecedented opportunities in cancerology. We applied independent component analysis (ICA) to bladder cancer transcriptome data sets and interpreted the components using gene enrichment analysis and tumor-associated molecular, clinicopathological, and processing information. We identified components associated with biological processes of tumor cells or the tumor microenvironment, and other components revealed technical biases. Applying ICA to nine cancer types identified cancer-shared and bladder-cancer-specific components. We characterized the luminal and basal-like subtypes of muscle-invasive bladder cancers according to the components identified. The study of the urothelial differentiation component, specific to the luminal subtypes, showed that a molecular urothelial differentiation program was maintained even in those luminal tumors that had lost morphological differentiation. Study of the genomic alterations associated with this component coupled with functional studies revealed a protumorigenic role for PPARG in luminal tumors. Our results support the inclusion of ICA in the exploitation of multiscale data sets. Graphical abstract Teaser Extracting biological insights from large-scale data is both a challenge and an opportunity. Biton et al. now analyze bladder tumor transcriptomes. An enrichment analysis of contributing genes combined with molecular and clinical annotations of tumor samples identifies biologically relevant components, some of which are shared with other carcinoma types.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Malgorzata Gozdecka , Stephen Lyons , Saki Kondo , Janet Taylor , Yaoyong Li , Jacek Walczynski , Gerald Thiel , Wolfgang Breitwieser , Nic Jones JNK and p38 phosphorylate a diverse set of substrates and, consequently, can act in a context-dependent manner to either promote or inhibit tumor growth. Elucidating the functions of specific substrates of JNK and p38 is therefore critical for our understanding of these kinases in cancer. ATF2 is a phosphorylation-dependent transcription factor and substrate of both JNK and p38. Here, we show ATF2 suppresses tumor formation in an orthotopic model of liver cancer and cellular transformation in vitro. Furthermore, we find that suppression of tumorigenesis by JNK requires ATF2. We identify a transcriptional program activated by JNK via ATF2 and provide examples of JNK- and ATF2-dependent genes that block cellular transformation. Significantly, we also show that ATF2-dependent gene expression is frequently downregulated in human cancers, indicating that amelioration of JNK-ATF2-mediated suppression may be a common event during tumor development. Graphical abstract Teaser Given the large number of substrates phosphorylated by stress-activated kinases, identifying key effectors of their antitumorigenic function is a major challenge. Gozdecka et al. provide evidence that ATF2 mediates the tumor-suppressive effect of JNK through a transcriptional program that is frequently downregulated in human tumors.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Shaheen Kabir , Dirk Hockemeyer , Titia de Lange The conserved protein Rap1 functions at telomeres in fungi, protozoa, and vertebrates. Like yeast Rap1, human Rap1 has been implicated in telomere length regulation and repression of nonhomologous end-joining (NHEJ) at telomeres. However, mouse telomeres lacking Rap1 do not succumb to NHEJ. To determine the functions of human Rap1, we generated several transcription activator-like effector nuclease (TALEN)-mediated human cell lines lacking Rap1. Loss of Rap1 did not affect the other components of shelterin, the modification of telomeric histones, the subnuclear position of telomeres, or the 3′ telomeric overhang. Telomeres lacking Rap1 did not show a DNA damage response, NHEJ, or consistent changes in their length, indicating that Rap1 does not have an important function in protection or length regulation of human telomeres. As human Rap1, like its mouse and unicellular orthologs, affects gene expression, we propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres. Graphical abstract Teaser Kabir et al. now employ a TALEN-mediated genome editing strategy to make Rap1 knockouts in a diverse array of human cell lines and test the function of this conserved telomere protein. They find that, unlike its yeast counterparts, mammalian Rap1 is not essential for telomere protection or length regulation, but its role in transcriptional regulation is maintained, indicating why Rap1 remains so conserved.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Carrie L. Simms , Benjamin H. Hudson , John W. Mosior , Ali S. Rangwala , Hani S. Zaher Chemical damage to RNA affects its functional properties and thus may pose a significant hurdle to the translational apparatus; however, the effects of damaged mRNA on the speed and accuracy of the decoding process and their interplay with quality-control processes are not known. Here, we systematically explore the effects of oxidative damage on the decoding process using a well-defined bacterial in vitro translation system. We find that the oxidative lesion 8-oxoguanosine (8-oxoG) reduces the rate of peptide-bond formation by more than three orders of magnitude independent of its position within the codon. Interestingly, 8-oxoG had little effect on the fidelity of the selection process, suggesting that the modification stalls the translational machinery. Consistent with these findings, 8-oxoG mRNAs were observed to accumulate and associate with polyribosomes in yeast strains in which no-go decay is compromised. Our data provide compelling evidence that mRNA-surveillance mechanisms have evolved to cope with damaged mRNA. Graphical abstract Teaser Oxidative damage to RNA has received relatively little attention despite evidence that it can accumulate in cells and is associated with numerous disease states. Simms et al. demonstrate that a single modified residue in an mRNA can lead to ribosomal stalling. Cells in which no-go decay is compromised show increased levels of 8-oxoG mRNA, suggesting that mRNA surveillance mechanisms may have evolved to cope with damaged mRNA.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Lawrence D. Gaspers , Paula J. Bartlett , Antonio Politi , Paul Burnett , Walson Metzger , Jane Johnston , Suresh K. Joseph , Thomas Höfer , Andrew P. Thomas Receptor-mediated oscillations in cytosolic Ca 2+ concentration ([Ca 2+ ] i ) could originate either directly from an autonomous Ca 2+ feedback oscillator at the inositol 1,4,5-trisphosphate (IP 3 ) receptor or as a secondary consequence of IP 3 oscillations driven by Ca 2+ feedback on IP 3 metabolism. It is challenging to discriminate these alternatives, because IP 3 fluctuations could drive Ca 2+ oscillations or could just be a secondary response to the [Ca 2+ ] i spikes. To investigate this problem, we constructed a recombinant IP 3 buffer using type-I IP 3 receptor ligand-binding domain fused to GFP (GFP-LBD), which buffers IP 3 in the physiological range. This IP 3 buffer slows hormone-induced [IP 3 ] dynamics without changing steady-state [IP 3 ]. GFP-LBD perturbed [Ca 2+ ] i oscillations in a dose-dependent manner: it decreased both the rate of [Ca 2+ ] i rise and the speed of Ca 2+ wave propagation and, at high levels, abolished [Ca 2+ ] i oscillations completely. These data, together with computational modeling, demonstrate that IP 3 dynamics play a fundamental role in generating [Ca 2+ ] i oscillations and waves. Graphical abstract Teaser Gaspers et al. use a genetically encoded IP 3 buffer to suppress IP 3 dynamics during hormonal stimulation. Using this approach, they find that positive feedback of Ca 2+ on IP 3 formation is an essential component, generating long-period, baseline-separated Ca 2+ oscillations and intracellular Ca 2+ waves.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Panos Roussos , Amanda C. Mitchell , Georgios Voloudakis , John F. Fullard , Venu M. Pothula , Jonathan Tsang , Eli A. Stahl , Anastasios Georgakopoulos , Douglas M. Ruderfer , Alexander Charney , Yukinori Okada , Katherine A. Siminovitch , Jane Worthington , Leonid Padyukov , Lars Klareskog , Peter K. Gregersen , Robert M. Plenge , Soumya Raychaudhuri , Menachem Fromer , Shaun M. Purcell , Kristen J. Brennand , Nikolaos K. Robakis , Eric E. Schadt , Schahram Akbarian , Pamela Sklar A large portion of common variant loci associated with genetic risk for schizophrenia reside within noncoding sequence of unknown function. Here, we demonstrate promoter and enhancer enrichment in schizophrenia variants associated with expression quantitative trait loci (eQTL). The enrichment is greater when functional annotations derived from the human brain are used relative to peripheral tissues. Regulatory trait concordance analysis ranked genes within schizophrenia genome-wide significant loci for a potential functional role, based on colocalization of a risk SNP, eQTL, and regulatory element sequence. We identified potential physical interactions of noncontiguous proximal and distal regulatory elements. This was verified in prefrontal cortex and -induced pluripotent stem cell–derived neurons for the L-type calcium channel ( CACNA1C ) risk locus. Our findings point to a functional link between schizophrenia-associated noncoding SNPs and 3D genome architecture associated with chromosomal loopings and transcriptional regulation in the brain. Graphical abstract Teaser Roussos et al. find that schizophrenia risk variants are enriched for alleles that affect gene expression and lie within promoters or enhancers. For the L-type calcium channel ( CACNA1C ), the risk variant is associated with transcriptional regulation in the brain and is positioned within an enhancer sequence that physically interacts though chromosome loops with the promoter region of the gene.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Denise Cook , Erin Nuro , Emma V. Jones , Haider F. Altimimi , W. Todd Farmer , Valentina Gandin , Edith Hanna , Ruiting Zong , Alessandro Barbon , David L. Nelson , Ivan Topisirovic , Joseph Rochford , David Stellwagen , Jean-Claude Béïque , Keith K. Murai Translational control of mRNAs allows for rapid and selective changes in synaptic protein expression that are required for long-lasting plasticity and memory formation in the brain. Fragile X Related Protein 1 (FXR1P) is an RNA-binding protein that controls mRNA translation in nonneuronal cells and colocalizes with translational machinery in neurons. However, its neuronal mRNA targets and role in the brain are unknown. Here, we demonstrate that removal of FXR1P from the forebrain of postnatal mice selectively enhances long-term storage of spatial memories, hippocampal late-phase long-term potentiation (L-LTP), and de novo GluA2 synthesis. Furthermore, FXR1P binds specifically to the 5′ UTR of GluA2 mRNA to repress translation and limit the amount of GluA2 that is incorporated at potentiated synapses. This study uncovers a mechanism for regulating long-lasting synaptic plasticity and spatial memory formation and reveals an unexpected divergent role of FXR1P among Fragile X proteins in brain plasticity. Graphical abstract Teaser Control over protein synthesis is important for long-lasting plasticity and memory storage in the brain. Cook, Nuro, et al. now reveal that the RNA-binding protein FXR1P acts as a molecular brake that limits synthesis and synaptic incorporation of the AMPAR subunit GluA2, ultimately constraining long-term plasticity and memory formation.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Fengtao Su , Shibani Mukherjee , Yanyong Yang , Eiichiro Mori , Souparno Bhattacharya , Junya Kobayashi , Steven M. Yannone , David J. Chen , Aroumougame Asaithamby WRN, the protein defective in Werner syndrome (WS), is a multifunctional nuclease involved in DNA damage repair, replication, and genome stability maintenance. It was assumed that the nuclease activities of WRN were critical for these functions. Here, we report a nonenzymatic role for WRN in preserving nascent DNA strands following replication stress. We found that lack of WRN led to shortening of nascent DNA strands after replication stress. Furthermore, we discovered that the exonuclease activity of MRE11 was responsible for the shortening of newly replicated DNA in the absence of WRN. Mechanistically, the N-terminal FHA domain of NBS1 recruits WRN to replication-associated DNA double-stranded breaks to stabilize Rad51 and to limit the nuclease activity of its C-terminal binding partner MRE11. Thus, this previously unrecognized nonenzymatic function of WRN in the stabilization of nascent DNA strands sheds light on the molecular reason for the origin of genome instability in WS individuals. Graphical abstract Teaser Su et al. uncover a nonenzymatic function for WRN in DNA replication, giving insight into the molecular origin of genome instability in Werner syndrome individuals. The authors find that WRN recruitment to replication-associated DNA double-strand breaks prevents excessive MRE11-mediated degradation of nascent DNA strands.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): David A. Gorczyca , Susan Younger , Shan Meltzer , Sung Eun Kim , Li Cheng , Wei Song , Hye Young Lee , Lily Yeh Jan , Yuh Nung Jan A major gap in our understanding of sensation is how a single sensory neuron can differentially respond to a multitude of different stimuli (polymodality), such as propio- or nocisensation. The prevailing hypothesis is that different stimuli are transduced through ion channels with diverse properties and subunit composition. In a screen for ion channel genes expressed in polymodal nociceptive neurons, we identified Ppk26, a member of the trimeric degenerin/epithelial sodium channel (DEG/ENaC) family, as being necessary for proper locomotion behavior in Drosophila larvae in a mutually dependent fashion with coexpressed Ppk1, another member of the same family. Mutants lacking Ppk1 and Ppk26 were defective in mechanical, but not thermal, nociception behavior. Mutants of Piezo, a channel involved in mechanical nociception in the same neurons, did not show a defect in locomotion, suggesting distinct molecular machinery for mediating locomotor feedback and mechanical nociception. Graphical abstract Teaser Using a screen for ion channels in polymodal nociceptive neurons, Gorczyca et al. identify Ppk26, a member of the trimeric DEG/ENaC channel family, as necessary for both proper locomotive behavior and mechanical, but not thermal, nociception.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Anindya Chatterjee , Joydeep Ghosh , Baskar Ramdas , Raghuveer Singh Mali , Holly Martin , Michihiro Kobayashi , Sasidhar Vemula , Victor H. Canela , Emily R. Waskow , Valeria Visconte , Ramon V. Tiu , Catherine C. Smith , Neil Shah , Kevin D. Bunting , H. Scott Boswell , Yan Liu , Rebecca J. Chan , Reuben Kapur Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs), and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK) whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis. Graphical abstract Teaser A significant impediment in treatment of leukemia, induced by oncogenic FLT3 and KIT receptors, is inadequate understanding of critical signaling pathways that lead to the development of this disease. In this study, Chatterjee et al. show an essential role of FAK/Tiam1/Rac1/PAK1 pathway in regulating nuclear translocation of Stat5 leading to leukemogenesis, in the context of oncogenic mutations of FLT3 and KIT, and provide multiple potential therapeutic targets to treat leukemia.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Shogo Tanabe , Toshihide Yamashita Multiple sclerosis (MS) is a chronic autoimmune disease characterized by inflammation, demyelination, and neurodegeneration in the CNS. Although it is important to prevent neurodegeneration for alleviating neurological disability, the molecular mechanism of neurodegeneration remains largely unknown. Here, we report that repulsive guidance molecule-a (RGMa), known to regulate axonal growth, is associated with neurodegeneration in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. RGMa is highly expressed in interleukin-17-producing CD4 + T cells (Th17 cells). We induced EAE by adoptive transfer of myelin oligodendrocyte glycoprotein (MOG)-specific Th17 cells and then inhibited RGMa with a neutralizing antibody. Inhibition of RGMa improves EAE scores and reduces neuronal degeneration without altering immune or glial responses. Th17 cells induce cultured cortical neuron death through RGMa-neogenin and Akt dephosphorylation. Our results demonstrate that RGMa is involved in Th17-cell-mediated neurodegeneration and that RGMa-specific antibody may have a therapeutic effect in MS. Graphical abstract Teaser The mechanism of neurodegeneration under inflammation in the CNS remains largely unknown. Tanabe and Yamashita demonstrate that RGMa is highly expressed in Th17 cells and induces dephosphorylation of Akt, leading to death of neurons. A neutralizing antibody to RGMa attenuates axonal degeneration and severity of Th17-cell-mediated autoimmune encephalomyelitis.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Patricia A. Possik , Judith Müller , Carmen Gerlach , Juliana C.N. Kenski , Xinyao Huang , Aida Shahrabi , Oscar Krijgsman , Ji-Ying Song , Marjon A. Smit , Bram Gerritsen , Cor Lieftink , Kristel Kemper , Magali Michaut , Roderick L. Beijersbergen , Lodewyk Wessels , Ton N. Schumacher , Daniel S. Peeper To identify factors preferentially necessary for driving tumor expansion, we performed parallel in vitro and in vivo negative-selection short hairpin RNA (shRNA) screens. Melanoma cells harboring shRNAs targeting several DNA damage response (DDR) kinases had a greater selective disadvantage in vivo than in vitro, indicating an essential contribution of these factors during tumor expansion. In growing tumors, DDR kinases were activated following hypoxia. Correspondingly, depletion or pharmacologic inhibition of DDR kinases was toxic to melanoma cells, including those that were resistant to BRAF inhibitor, and this could be enhanced by angiogenesis blockade. These results reveal that hypoxia sensitizes melanomas to targeted inhibition of the DDR and illustrate the utility of in vivo shRNA dropout screens for the identification of pharmacologically tractable targets. Graphical abstract Teaser Specific parameters lacking in vitro, but present in vivo, influence tumor behavior and therapeutic dependencies. Possik et al. now perform parallel in vitro and in vivo negative-selection screens and uncover a critical requirement for DNA damage response kinases in expanding tumors, a liability that could be exploited pharmacologically.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Debabrata Panja , Justin W. Kenney , Laura D’Andrea , Francesca Zalfa , Anni Vedeler , Karin Wibrand , Rikiro Fukunaga , Claudia Bagni , Christopher G. Proud , Clive R. Bramham BDNF signaling contributes to protein-synthesis-dependent synaptic plasticity, but the dynamics of TrkB signaling and mechanisms of translation have not been defined. Here, we show that long-term potentiation (LTP) consolidation in the dentate gyrus of live rodents requires sustained (hours) BDNF-TrkB signaling. Surprisingly, this sustained activation maintains an otherwise labile signaling pathway from TrkB to MAP-kinase-interacting kinase (MNK). MNK activity promotes eIF4F translation initiation complex formation and protein synthesis in mechanistically distinct early and late stages. In early-stage translation, MNK triggers release of the CYFIP1/FMRP repressor complex from the 5′-mRNA cap. In late-stage translation, MNK regulates the canonical translational repressor 4E-BP2 in a synapse-compartment-specific manner. This late stage is coupled to MNK-dependent enhanced dendritic mRNA translation. We conclude that LTP consolidation in the dentate gyrus is mediated by sustained BDNF signaling to MNK and MNK-dependent regulation of translation in two functionally and mechanistically distinct stages. Graphical abstract Teaser The logic of translational control in synaptic plasticity is not well understood. Panja et al. show that long-term potentiation in the dentate gyrus of live rodents is a two-stage process driven by brain-derived neurotrophic factor signaling to MAP-kinase-interacting kinase and activation of functionally and mechanistically distinct forms of translation.

Description: Publication date: Available online 16 January 2014 Source: Cell Reports Author(s): Xiaqin Sun , Yu Wu , Mingxue Gu , Yan Zhang MicroRNA alterations and axonopathy have been reported in patients with Alzheimer’s disease (AD) and in AD mouse models. We now report that miR-342-5p is upregulated in APP/PS1, PS1ΔE9, and PS1-M146V transgenic AD mice, and that this upregulation is mechanistically linked to elevated β-catenin, c-Myc, and interferon regulatory factor-9. The increased miR-342-5p downregulates the expression of ankyrin G (AnkG), a protein that is known to play a critical role at the axon initial segment. Thus, a specific miRNA alteration may contribute to AD axonopathy by downregulating AnkG. Graphical abstract Teaser MicroRNA (miRNA) alterations and axonopathy have been reported in patients with Alzheimer’s disease (AD) and AD mouse models. In this study, Zhang and colleagues show that miR-342-5p is upregulated in APP/PS1, PS1ΔE9, and PS1-M146V transgenic AD mice. The increased miR-342-5p downregulates the expression of ankyrin G (AnkG), a protein that is known to play a critical role at the axon initial segment. Thus, a specific miRNA alteration may contribute to AD axonopathy by downregulating AnkG.

Description: Publication date: Available online 16 January 2014 Source: Cell Reports Author(s): Filip Janku , David S. Hong , Siqing Fu , Sarina A. Piha-Paul , Aung Naing , Gerald S. Falchook , Apostolia M. Tsimberidou , Vanda M. Stepanek , Stacy L. Moulder , J. Jack Lee , Rajyalakshmi Luthra , Ralph G. Zinner , Russell R. Broaddus , Jennifer J. Wheler , Razelle Kurzrock Despite a wealth of preclinical studies, it is unclear whether PIK3CA or phosphatase and tensin homolog (PTEN) gene aberrations are actionable in the clinical setting. Of 1,656 patients with advanced, refractory cancers tested for PIK3CA or PTEN abnormalities, PIK3CA mutations were found in 9% (146/1,589), and PTEN loss and/or mutation was found in 13% (149/1,157). In multicovariable analysis, treatment with a phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) inhibitor was the only independent factor predicting response to therapy in individuals harboring a PIK3CA or PTEN aberration. The rate of stable disease ≥6 months/partial response reached 45% in a subgroup of individuals with H1047R PIK3CA mutations. Aberrations in the PI3K/AKT/mTOR pathway are common and potentially actionable in patients with diverse advanced cancers. This work provides further important clinical validation for continued and accelerated use of biomarker-driven trials incorporating rational drug combinations. Graphical abstract Teaser Despite a wealth of preclinical studies, it is unclear whether PIK3CA or PTEN gene aberrations are actionable in the clinical setting. Janku and colleagues show that, even in patients with advanced refractory tumors, treatment with PI3K/AKT/mTOR axis inhibitors can produce significant responses and that these responses are observed across multiple histologies. Furthermore, patients whose tumors harbor an H1047R PIK3CA mutation may do especially well when cognate inhibitors are given. This work has important implications for clinical management.

Description: Publication date: Available online 16 January 2014 Source: Cell Reports Author(s): Matthew L. Bochman , Katrin Paeschke , Angela Chan , Virginia A. Zakian Human RecQ4 (hRecQ4) affects cancer and aging but is difficult to study because it is a fusion between a helicase and an essential replication factor. Budding yeast Hrq1 is homologous to the disease-linked helicase domain of RecQ4 and, like hRecQ4, is a robust 3′-5′ helicase. Additionally, Hrq1 has the unusual property of forming heptameric rings. Cells lacking Hrq1 exhibited two DNA damage phenotypes: hypersensitivity to DNA interstrand crosslinks (ICLs) and telomere addition to DNA breaks. Both activities are rare; their coexistence in a single protein is unprecedented. Resistance to ICLs requires helicase activity, but suppression of telomere addition does not. Hrq1 also affects telomere length by a noncatalytic mechanism, as well as telomerase-independent telomere maintenance. Because Hrq1 binds telomeres in vivo, it probably affects them directly. Thus, the tumor-suppressing activity of RecQ4 could be due to a role in ICL repair and/or suppression of de novo telomere addition. Graphical abstract Teaser In this study, Bochman and colleagues show that, in vitro, the Saccharomyces cerevisiae Hrq1 is a functional homolog of the disease-linked human RecQ4 helicase, establishing Hrq1 as the second RecQ helicase in budding yeast and a simple model for RecQ4 studies. They go on to show that Hrq1 functions catalytically during DNA interstrand crosslink repair and structurally to prevent telomere addition to DNA double-strand breaks and chromosome ends.

Description: Publication date: Available online 16 January 2014 Source: Cell Reports Author(s): Michael S. Minett , Sarah Falk , Sonia Santana-Varela , Yury D. Bogdanov , Mohammed A. Nassar , Anne-Marie Heegaard , John N. Wood Nav1.7, a peripheral neuron voltage-gated sodium channel, is essential for pain and olfaction in mice and humans. We examined the role of Nav1.7 as well as Nav1.3, Nav1.8, and Nav1.9 in different mouse models of chronic pain. Constriction-injury-dependent neuropathic pain is abolished when Nav1.7 is deleted in sensory neurons, unlike nerve-transection-related pain, which requires the deletion of Nav1.7 in sensory and sympathetic neurons for pain relief. Sympathetic sprouting that develops in parallel with nerve-transection pain depends on the presence of Nav1.7 in sympathetic neurons. Mechanical and cold allodynia required distinct sets of neurons and different repertoires of sodium channels depending on the nerve injury model. Surprisingly, pain induced by the chemotherapeutic agent oxaliplatin and cancer-induced bone pain do not require the presence of Nav1.7 sodium channels or Nav1.8-positive nociceptors. Thus, similar pain phenotypes arise through distinct cellular and molecular mechanisms. Therefore, rational analgesic drug therapy requires patient stratification in terms of mechanisms and not just phenotype. Graphical abstract Teaser Wood and colleagues describe two pain syndromes that occur in the absence of Nav1.7, a sodium channel considered to be essential for pain perception and olfaction in humans. They provide evidence that pain phenotypes such as cold and mechanical allodynia can arise through distinct cell and molecular mechanisms after nerve injury in mouse peripheral sensory neurons. The existence of redundant mechanistically distinct peripheral pain mechanisms may help to explain recent difficulties with the development of new analgesic drugs.

Description: Publication date: Available online 16 January 2014 Source: Cell Reports Author(s): Yilin Tai , Justyna A. Janas , Chia-Lin Wang , Linda Van Aelst Chandelier cells (ChCs), typified by their unique axonal morphology, are the most distinct interneurons present in cortical circuits. Via their distinctive axonal terminals, called cartridges, these cells selectively target the axon initial segment of pyramidal cells and control action potential initiation; however, the mechanisms that govern the characteristic ChC axonal structure have remained elusive. Here, by employing an in utero electroporation-based method that enables genetic labeling and manipulation of ChCs in vivo, we identify DOCK7, a member of the DOCK180 family, as a molecule essential for ChC cartridge and bouton development. Furthermore, we present evidence that DOCK7 functions as a cytoplasmic activator of the schizophrenia-associated ErbB4 receptor tyrosine kinase and that DOCK7 modulates ErbB4 activity to control ChC cartridge and bouton development. Thus, our findings define DOCK7 and ErbB4 as key components of a pathway that controls the morphological differentiation of ChCs, with implications for the pathogenesis of schizophrenia. Graphical abstract Teaser Chandelier cells (ChCs), typified by their unique axonal morphology, are the most distinct interneurons present in cortical circuits; however, the mechanisms that govern their characteristic axonal structure have remained elusive. In this study, Van Aelst and colleagues describe an in utero electroporation-based method that enables genetic labeling and manipulation of ChCs in vivo. Using this approach, they uncover a critical role for DOCK7 as a cytoplasmic activator of receptor tyrosine kinase ErbB4 in the regulation of ChC cartridge and bouton development.

Description: Publication date: Available online 16 January 2014 Source: Cell Reports Author(s): Patricia Heyn , Martin Kircher , Andreas Dahl , Janet Kelso , Pavel Tomancak , Alex T. Kalinka , Karla M. Neugebauer The transition from maternal to zygotic control is fundamental to the life cycle of all multicellular organisms. It is widely believed that genomes are transcriptionally inactive from fertilization until zygotic genome activation (ZGA). Thus, the earliest genes expressed probably support the rapid cell divisions that precede morphogenesis and, if so, might be evolutionarily conserved. Here, we identify the earliest zygotic transcripts in the zebrafish, Danio rerio , through metabolic labeling and purification of RNA from staged embryos. Surprisingly, the mitochondrial genome was highly active from the one-cell stage onwards, showing that significant transcriptional activity exists at fertilization. We show that 592 nuclear genes become active when cell cycles are still only 15 min long, confining expression to relatively short genes. Furthermore, these zygotic genes are evolutionarily younger than those expressed at other developmental stages. Comparison of fish, fly, and mouse data revealed different sets of genes expressed at ZGA. This species specificity uncovers an evolutionary plasticity in early embryogenesis that probably confers substantial adaptive potential. Graphical abstract Teaser The development of all metazoans is initially controlled by maternal factors, and transcription of the zygotic genome starts only later. In this study, Tomancak, Neugebauer, and colleagues identify the first zygotic transcripts in zebrafish with a 4-sUTP metabolic labeling approach. Using this data set, they find that the first zygotic transcripts in different species are not shared, whereas the maternally deposited RNAs are highly enriched for shared orthologs. These results highlight the striking flexibility of early development.

Description: Publication date: Available online 16 January 2014 Source: Cell Reports Author(s): Yakov Chudnovsky , Dohoon Kim , Siyuan Zheng , Warren A. Whyte , Mukesh Bansal , Mark-Anthony Bray , Shuba Gopal , Matthew A. Theisen , Steve Bilodeau , Prathapan Thiru , Julien Muffat , Omer H. Yilmaz , Maya Mitalipova , Kevin Woolard , Jeongwu Lee , Riko Nishimura , Nobuo Sakata , Howard A. Fine , Anne E. Carpenter , Serena J. Silver , Roel G.W. Verhaak , Andrea Califano , Richard A. Young , Keith L. Ligon , Ingo K. Mellinghoff , David E. Root , David M. Sabatini , William C. Hahn , Milan G. Chheda Glioblastoma (GBM) harbors subpopulations of therapy-resistant tumor-initiating cells (TICs) that are self-renewing and multipotent. To understand the regulation of the TIC state, we performed an image-based screen for genes regulating GBM TIC maintenance and identified ZFHX4 , a 397 kDa transcription factor. ZFHX4 is required to maintain TIC-associated and normal human neural precursor cell phenotypes in vitro, suggesting that ZFHX4 regulates differentiation, and its suppression increases glioma-free survival in intracranial xenografts. ZFHX4 interacts with CHD4, a core member of the nucleosome remodeling and deacetylase (NuRD) complex. ZFHX4 and CHD4 bind to overlapping sets of genomic loci and control similar gene expression programs. Using expression data derived from GBM patients, we found that ZFHX4 significantly affects CHD4-mediated gene expression perturbations, which defines ZFHX4 as a master regulator of CHD4. These observations define ZFHX4 as a regulatory factor that links the chromatin-remodeling NuRD complex and the GBM TIC state. Graphical abstract Teaser Glioblastoma (GBM), the most common and aggressive primary brain tumor, contains a subpopulation of stem cell-like, therapy-resistant tumor-initiating cells (TICs). Sabatini, Hahn, and colleagues performed an image-based RNAi screen in order to identify candidate regulators of GBM TIC functions. ZFHX4, identified in this screen, is essential for the stem cell-like state and tumorigenicity of TICs. Additionally, ZFHX4 interacts with CHD4, a core member of the chromatin regulatory NuRD complex, and drives CHD4-dependent gene expression programs.

Description: Publication date: Available online 13 March 2014 Source: Cell Reports Author(s): Saul J. Priceman , Shudan Shen , Lin Wang , Jiehui Deng , Chanyu Yue , Maciej Kujawski , Hua Yu S1PR1 signaling has been shown to restrain the number and function of regulatory T (Treg) cells in the periphery under physiological conditions and in colitis models, but its role in regulating tumor-associated T cells is unknown. Here, we show that S1PR1 signaling in T cells drives Treg accumulation in tumors, limits CD8 + T cell recruitment and activation, and promotes tumor growth. T-cell-intrinsic S1PR1 affects Treg cells, but not CD8 + T cells, as demonstrated by adoptive transfer models and transient pharmacological S1PR1 modulation. An increase in S1PR1 in CD4 + T cells promotes STAT3 activation and JAK/STAT3-dependent Treg tumor migration, whereas STAT3 ablation in T cells diminishes tumor-associated Treg accumulation and tumor growth. Our study demonstrates a stark contrast between the consequences of S1PR1 signaling in Treg cells in the periphery versus tumors. Graphical abstract Teaser Although SIPR1 restrains regulatory T (Treg) cells in the periphery in naive and some inflammatory conditions, its impact on Treg cells in tumors has not yet been explored. In the current study, Yu and colleagues show that S1PR1 signaling, via JAK/STAT3 activation in T cells, drives tumor accumulation of Treg cells, limits CD8 + T cell recruitment and activation, and promotes solid tumor growth. These studies highlight a potential T cell target for future cancer immunotherapy.

Description: Publication date: 13 March 2014 Source: Cell Reports, Volume 6, Issue 5 Author(s): William C. Hines , Ying Su , Irene Kuhn , Kornelia Polyak , Mina J. Bissell

Description: Publication date: Available online 13 March 2014 Source: Cell Reports Author(s): Manjula Pandey , Smita S. Patel By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading-strand synthesis, taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound/copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without guanine:cytosine (GC) dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate, whereas the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a model in which the helicase and polymerase are moving in one-nucleotide steps, DNA synthesis drives fork unwinding, and a role of the helicase is to trap the unwound bases and prevent DNA reannealing. Graphical abstract Teaser Pandey and Patel now show that a replicative helicase and polymerase increase each other’s rates of unwinding and synthesis. Aiming to understand the mechanism of cooperative catalysis, the authors show that the helicase and polymerase are located in close proximity to the fork junction and advance the replication fork synchronously, taking single-nucleotide steps coupled to hydrolysis of one nucleotide triphosphate. They propose that DNA synthesis drives unwinding and that the helicase aids this process by base trapping.

Description: Publication date: Available online 13 March 2014 Source: Cell Reports Author(s): Chieh-Yang Cheng , Chang-Il Hwang , David C. Corney , Andrea Flesken-Nikitin , Longchang Jiang , Gülfem Meryem Öner , Robert J. Munroe , John C. Schimenti , Heiko Hermeking , Alexander Yu. Nikitin The miR-34 family was originally found to be a direct target of p53 and is a group of putative tumor suppressors. Surprisingly, mice lacking all mir-34 genes show no increase in cancer formation by 18 months of age, hence placing the physiological relevance of previous studies in doubt. Here, we report that mice with prostate epithelium-specific inactivation of mir-34 and p53 show expansion of the prostate stem cell compartment and develop early invasive adenocarcinomas and high-grade prostatic intraepithelial neoplasia, whereas no such lesions are observed after inactivation of either the mir-34 or p53 genes alone by 15 months of age. Consistently, combined deficiency of p53 and miR-34 leads to acceleration of MET-dependent growth, self-renewal, and motility of prostate stem/progenitor cells. Our study provides direct genetic evidence that mir-34 genes are bona fide tumor suppressors and identifies joint control of MET expression by p53 and miR-34 as a key component of prostate stem cell compartment regulation, aberrations in which may lead to cancer. Graphical abstract Teaser MicroRNAs of the miR-34 family were originally identified as downstream effectors of tumor suppressor gene p53 . However, their presumptive tumor-suppressive role was placed in doubt based on studies of mice lacking mir-34 genes. By using mice with prostate-specific gene inactivation, Nikitin and colleagues show that miR-34 cooperates with p53 in suppression of prostate cancer. The lack of both miR-34 and p53 results in increased expression of MET receptor tyrosine kinase, which is responsible for expansion of the cancer-prone prostate stem cell compartment.

Description: Publication date: Available online 13 March 2014 Source: Cell Reports Author(s): Wenqiang Liu , Jiqing Yin , Xiaochen Kou , Yonghua Jiang , Haibo Gao , Yanhong Zhao , Bo Huang , Wenteng He , Hong Wang , Zhiming Han , Shaorong Gao It has been demonstrated that reprogramming factors are sequestered in the pronuclei of zygotes after fertilization, because zygotes enucleated at the M phase instead of interphase of the first mitosis can support the development of cloned embryos. However, the contribution of the parental pronucleus derived from either the sperm or the oocyte in reprogramming remains elusive. Here, we demonstrate that the parental pronuclei have asymmetric reprogramming capacities and that the reprogramming factors reside predominantly in the male pronucleus. As a result, only female pronucleus-depleted (FPD) mouse zygotes can reprogram somatic cells to a pluripotent state and support the full-term development of cloned embryos; male pronucleus-depleted (MPD) zygotes fail to support somatic cell reprogramming. We further demonstrate that fusion of an additional male pronucleus into a zygote greatly enhances reprogramming efficiency. Our data provide a clue to further identify critical reprogramming factors in the male pronucleus. Graphical abstract Teaser Somatic cell nuclear transfer (SCNT) experiments have shown that oocytes in metaphase II can reprogram somatic cells. Han, Gao, and colleagues now demonstrate that reprogramming factors are sequestered predominantly in the male pronucleus following fertilization, and that female pronucleus-depleted zygotes can reprogram somatic cells to pluripotency, whereas male pronucleus-depleted (MPD) zygotes fail to support somatic cell reprogramming.

Description: Publication date: Available online 13 March 2014 Source: Cell Reports Author(s): Bénédicte F. Py , Mingzhi Jin , Bimal N. Desai , Anirudh Penumaka , Hong Zhu , Maike Kober , Alexander Dietrich , Marta M. Lipinski , Thomas Henry , David E. Clapham , Junying Yuan Caspase-11 is a highly inducible caspase that controls both inflammatory responses and cell death. Caspase-11 controls interleukin 1β (IL-1β) secretion by potentiating caspase-1 activation and induces caspase-1-independent pyroptosis downstream of noncanonical NLRP3 inflammasome activators such as lipopolysaccharide (LPS) and Gram-negative bacteria. However, we still know very little about the downstream mechanism of caspase-11 in regulating inflammation because the known substrates of caspase-11 are only other caspases. Here, we identify the cationic channel subunit transient receptor potential channel 1 (TRPC1) as a substrate of caspase-11. TRPC1 deficiency increases the secretion of IL-1β without modulating caspase-1 cleavage or cell death in cultured macrophages. Consistently, trpc1 −/− mice show higher IL-1β secretion in the sepsis model of intraperitoneal LPS injection. Altogether, our data suggest that caspase-11 modulates the cationic channel composition of the cell and thus regulates the unconventional secretion pathway in a manner independent of caspase-1. Graphical abstract Teaser Caspase-11 is a critical proinflammatory caspase that can promote caspase-1 activation, pro-IL1β cleavage, and caspase-3 activation to mediate apoptosis under inflammatory conditions. However, the mechanism by which caspase-11 mediates inflammation is not clear because the only known substrates of caspase-11 are other caspases. Here, Yuan and colleagues identify the cationic channel subunit TRPC1 as a noncaspase substrate of caspase-11 and demonstrate a mechanism by which caspase-11 regulates the unconventional secretion of IL-1β by mediating the degradation of TRPC1.

Description: Publication date: Available online 13 March 2014 Source: Cell Reports Author(s): Megan R. Edwards , Britney Johnson , Chad E. Mire , Wei Xu , Reed S. Shabman , Lauren N. Speller , Daisy W. Leung , Thomas W. Geisbert , Gaya K. Amarasinghe , Christopher F. Basler Kelch-like ECH-associated protein 1 (Keap1) is a ubiquitin E3 ligase specificity factor that targets transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) for ubiquitination and degradation. Disrupting Keap1-Nrf2 interaction stabilizes Nrf2, resulting in Nrf2 nuclear accumulation, binding to antioxidant response elements (AREs), and transcription of cytoprotective genes. Marburg virus (MARV) is a zoonotic pathogen that likely uses bats as reservoir hosts. We demonstrate that MARV protein VP24 (mVP24) binds the Kelch domain of either human or bat Keap1. This binding is of high affinity and 1:1 stoichiometry and activates Nrf2. Modeling based on the Zaire ebolavirus (EBOV) VP24 (eVP24) structure identified in mVP24 an acidic loop (K-loop) critical for Keap1 interaction. Transfer of the K-loop to eVP24, which otherwise does not bind Keap1, confers Keap1 binding and Nrf2 activation, and infection by MARV, but not EBOV, activates ARE gene expression. Therefore, MARV targets Keap1 to activate Nrf2-induced cytoprotective responses during infection. Graphical abstract Teaser The antioxidant response is increasingly recognized to impact viral infections. Cellular protein Keap1 is a negative regulator of transcription factor Nrf2, master regulator of the antioxidant response. In this study, Basler and colleagues show that the VP24 protein of the highly lethal Marburg virus (MARV) directly interacts with the Kelch domain of Keap1. This activates Nrf2-dependent antioxidant gene expression in MARV-infected cells. These results highlight the potential for MARV to modulate host antioxidant responses to enhance virus replication.

Description: Publication date: Available online 13 March 2014 Source: Cell Reports Author(s): Wendan Ren , Hongxia Chen , Qiangzu Sun , Xuhua Tang , Siew Choo Lim , Jun Huang , Haiwei Song The SOSS1 complex comprising SOSSA, SOSSB1, and SOSSC senses single-stranded DNA (ssDNA) and promotes repair of DNA double-strand breaks (DSBs). But how SOSS1 is assembled and recognizes ssDNA remains elusive. The crystal structure of the N-terminal half of SOSSA (SOSSA N ) in complex with SOSSB1 and SOSSC showed that SOSSA N serves as a scaffold to bind both SOSSB1 and SOSSC for assembly of the SOSS1 complex. The structures of SOSSA N /B1 in complex with a 12 nt ssDNA and SOSSA N /B1/C in complex with a 35 nt ssDNA showed that SOSSB1 interacts with both SOSSA N and ssDNA via two distinct surfaces. Recognition of ssDNA with a length of up to nine nucleotides is mediated solely by SOSSB1, whereas neither SOSSC nor SOSSA N are critical for ssDNA binding. These results reveal the structural basis of SOSS1 assembly and provide a framework for further study of the mechanism governing longer ssDNA recognition by the SOSS1 complex during DSB repair. Graphical abstract Teaser The SOSS1 complex senses single-stranded DNA and promotes repair of DNA double-strand breaks (DSBs). Huang, Song, and colleagues have now determined the crystal structures of a truncated form of SOSS1 in isolation and in complex with single-stranded DNA (ssDNA). They show that SOSSA acts as a scaffold for SOSS1 assembly and that SOSSB1 binds to SOSSA and ssDNA through two distinct surfaces. These findings reveal the structural basis of SOSS1 assembly and provide a framework for further elucidating the role of SOSS1 in DSB repair.

Description: Publication date: Available online 13 March 2014 Source: Cell Reports Author(s): Audrey Page , Valentina A. Volchkova , Saint Patrick Reid , Mathieu Mateo , Audrey Bagnaud-Baule , Kirill Nemirov , Amy C. Shurtleff , Philip Lawrence , Oliver Reynard , Michele Ottmann , Vincent Lotteau , Shyam S. Biswal , Rajesh K. Thimmulappa , Sina Bavari , Viktor E. Volchkov Marburg virus (MARV) has a high fatality rate in humans, causing hemorrhagic fever characterized by massive viral replication and dysregulated inflammation. Here, we demonstrate that VP24 of MARV binds Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of nuclear transcription factor erythroid-derived 2 (Nrf2). Binding of VP24 to Keap1 Kelch domain releases Nrf2 from Keap1-mediated inhibition promoting persistent activation of a panoply of cytoprotective genes implicated in cellular responses to oxidative stress and regulation of inflammatory responses. Increased expression of Nrf2-dependent genes was demonstrated both during MARV infection and upon ectopic expression of MARV VP24. We also show that Nrf2-deficient mice can control MARV infection when compared to lethal infection in wild-type animals, indicating that Nrf2 is critical for MARV infection. We conclude that VP24-driven activation of the Nrf2-dependent pathway is likely to contribute to dysregulation of host antiviral inflammatory responses and that it ensures survival of MARV-infected cells despite these responses. Graphical abstract Teaser The Nrf2/Keap1-dependent cytoprotective pathway plays an important role in regulation of cellular responses to oxidative stress and inflammation. Keap1 controls Nrf2 transcriptional activity by targeting this protein for proteosomal degradation. Volchkov and colleagues now demonstrate that Marburgvirus hijacks this pathway via MARV VP24 binding to Keap1 that liberates Nrf2 from Keap1 control. MARV causes sustained and uncontrolled Nrf2 pathway activation that ensures cytoprotection of infected cells and contributes to dysregulation of host inflammatory responses to infection and high pathogenicity.

Description: Publication date: Available online 13 March 2014 Source: Cell Reports Author(s): Anna-Karin E. Johnsson , Yanfeng Dai , Max Nobis , Martin J. Baker , Ewan J. McGhee , Simon Walker , Juliane P. Schwarz , Shereen Kadir , Jennifer P. Morton , Kevin B. Myant , David J. Huels , Anne Segonds-Pichon , Owen J. Sansom , Kurt I. Anderson , Paul Timpson , Heidi C.E. Welch The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments. Graphical abstract Teaser The small G protein Rac is a signaling switch that controls cell morphology and migration. Here, Timpson, Welch, and colleagues present a mouse strain, Rac-FRET, which enables the imaging and quantification of Rac activity in living tissue and primary cells. Their use of the Rac-FRET mouse reveals novel patterns of Rac activity in neutrophil chemotaxis and unexpected insights into Rac signaling in normal or disease states of the intestine, liver, mammary tissue, pancreas, and skin.

Description: Publication date: Available online 13 March 2014 Source: Cell Reports Author(s): Salman Syed , Manjula Pandey , Smita S. Patel , Taekjip Ha Bacteriophage T7 gp4 serves as a model protein for replicative helicases that couples deoxythymidine triphosphate (dTTP) hydrolysis to directional movement and DNA strand separation. We employed single-molecule fluorescence resonance energy transfer methods to resolve steps during DNA unwinding by T7 helicase. We confirm that the unwinding rate of T7 helicase decreases with increasing base pair stability. For duplexes containing >35% guanine-cytosine (GC) base pairs, we observed stochastic pauses every 2–3 bp during unwinding. The dwells on each pause were distributed nonexponentially, consistent with two or three rounds of dTTP hydrolysis before each unwinding step. Moreover, we observed backward movements of the enzyme on GC-rich DNAs at low dTTP concentrations. Our data suggest a coupling ratio of 1:1 between base pairs unwound and dTTP hydrolysis, and they further support the concept that nucleic acid motors can have a hierarchy of different-sized steps or can accumulate elastic energy before transitioning to a subsequent phase. Graphical abstract Teaser Ha and colleagues provide direct evidence that the T7 helicase unwinds DNA in discrete steps of 2–3 bp. Each unwinding step consists of two or three hidden steps, suggesting a coupling ratio of 1:1 between base pairs unwound and dNTP hydrolyzed, and indicating accumulation of elastic energy, before transitioning to a subsequent phase. At low dNTP, the helicase also displays backward movements, likely due to loss of subunit coordination.

Description: Publication date: Available online 1 May 2014 Source: Cell Reports Author(s): Timothy O’Sullivan , Robert Saddawi-Konefka , Emilie Gross , Miller Tran , Stephen P. Mayfield , Hiroaki Ikeda , Jack D. Bui The process of cancer immunoediting generates a repertoire of cancer cells that can persist in immune-competent hosts. In its most complex form, this process begins with the elimination of highly immunogenic unedited tumor cells followed by the escape of less immunogenic edited cells. Although edited tumors can release immunosuppressive factors, it is unknown whether unedited tumors produce cytokines that enhance antitumor function. Utilizing gene microarray analysis, we found the cytokine interleukin 17D (IL-17D) was highly expressed in certain unedited tumors but not in edited mouse tumor cell lines. Moreover, forced expression of IL-17D in edited tumor cells induced rejection by stimulating MCP-1 production from tumor endothelial cells, leading to the recruitment of natural killer (NK) cells. NK cells promoted M1 macrophage development and adaptive immune responses. IL-17D expression was also decreased in certain high-grade and metastatic human tumors, suggesting that it can be targeted for tumor immune therapy. Graphical abstract Teaser Cytokines are used by the immune system to regulate inflammation and host responses. Certain cytokines can be expressed outside of the immune system, presumably by tissues, to initiate immune surveillance pathways. Herein, O’Sullivan et al. show that the cytokine interleukin-17D (IL-17D) is expressed by immunogenic tumor cells. IL-17D acts by inducing MCP-1 and recruiting natural killer cells, leading to tumor destruction. These results imply that IL-17D may be useful in cancer immune therapy.

Description: Publication date: Available online 1 May 2014 Source: Cell Reports Author(s): Luigi Mari , Francesca Milano , Kaushal Parikh , Danielle Straub , Vincent Everts , Kees K. Hoeben , Paul Fockens , Navtej S. Buttar , Kausilia K. Krishnadath The molecular mechanisms leading to epithelial metaplasias are poorly understood. Barrett's esophagus is a premalignant metaplastic change of the esophageal epithelium into columnar epithelium, occurring in patients suffering from gastroesophageal reflux disease. Mechanisms behind the development of the intestinal subtype, which is associated with the highest cancer risk, are unclear. In humans, it has been suggested that a nonspecialized columnar metaplasia precedes the development of intestinal metaplasia. Here, we propose that a complex made up of at least two factors needs to be activated simultaneously to drive the expression of intestinal type of genes. Using unique animal models and robust in vitro assays, we show that the nonspecialized columnar metaplasia is a precursor of intestinal metaplasia and that pSMAD/CDX2 interaction is essential for the switch toward an intestinal phenotype. Graphical abstract Teaser The molecular mechanisms leading to the preneoplastic intestinal type esophageal metaplasia seen in Barrett’s esophagus are unclear. Using unique animal models and robust in vitro assays, Mari et al. show that a nonspecialized columnar metaplasia is the precursor of intestinal metaplasia and that a collaboration between the transcriptional factors CDX-2 and pSMAD is essential for the transformation into the intestinal phenotype. This concept has far-reaching implications, given that the development of intestinal metaplasia predisposes for several highly malignant cancers.

Description: Publication date: Available online 1 May 2014 Source: Cell Reports Author(s): Olga Murina , Christine von Aesch , Ufuk Karakus , Lorenza P. Ferretti , Hella A. Bolck , Kay Hänggi , Alessandro A. Sartori The resolution of DNA interstrand crosslinks (ICLs) requires a complex interplay between several processes of DNA metabolism, including the Fanconi anemia (FA) pathway and homologous recombination (HR). FANCD2 monoubiquitination and CtIP-dependent DNA-end resection represent key events in FA and HR activation, respectively, but very little is known about their functional relationship. Here, we show that CtIP physically interacts with both FANCD2 and ubiquitin and that monoubiquitinated FANCD2 tethers CtIP to damaged chromatin, which helps channel DNA double-strand breaks generated during ICL processing into the HR pathway. Consequently, CtIP mutants defective in FANCD2 binding fail to associate with damaged chromatin, which leads to increased levels of nonhomologous end-joining activity and ICL hypersensitivity. Interestingly, we also observe that CtIP depletion aggravates the genomic instability in FANCD2-deficient cells. Thus, our data indicate that FANCD2 primes CtIP-dependent resection during HR after ICL induction but that CtIP helps prevent illegitimate recombination in FA cells. Graphical abstract Teaser In response to DNA interstrand crosslinks (ICLs), FANCD2 coordinates several DNA repair mechanisms in order to promote genome stability, yet its connection to homologous recombination (HR) is poorly understood. Here, Murina et al. show that FANCD2 primes CtIP-mediated DNA end resection, thereby promoting error-free HR during ICL repair. They show that CtIP is tethered to damaged chromatin by physically interacting with FANCD2 and most likely by its ability to recognize ubiquitinated substrates.

Description: Publication date: Available online 1 May 2014 Source: Cell Reports Author(s): William R. Harcombe , William J. Riehl , Ilija Dukovski , Brian R. Granger , Alex Betts , Alex H. Lang , Gracia Bonilla , Amrita Kar , Nicholas Leiby , Pankaj Mehta , Christopher J. Marx , Daniel Segrè The interspecies exchange of metabolites plays a key role in the spatiotemporal dynamics of microbial communities. This raises the question of whether ecosystem-level behavior of structured communities can be predicted using genome-scale metabolic models for multiple organisms. We developed a modeling framework that integrates dynamic flux balance analysis with diffusion on a lattice and applied it to engineered communities. First, we predicted and experimentally confirmed the species ratio to which a two-species mutualistic consortium converges and the equilibrium composition of a newly engineered three-member community. We next identified a specific spatial arrangement of colonies, which gives rise to what we term the “eclipse dilemma”: does a competitor placed between a colony and its cross-feeding partner benefit or hurt growth of the original colony? Our experimentally validated finding that the net outcome is beneficial highlights the complex nature of metabolic interactions in microbial communities while at the same time demonstrating their predictability. Graphical abstract Teaser Microbes can interact with the environment and with each other through the uptake and secretion of metabolites. Here, Harcombe et al. ask whether mathematical modeling of the metabolic network of individual species can help forecast the spatiotemporal behavior of two- and three-species engineered microbial ecosystems. In addition to accurately predicting colony growth rates and equilibrium community compositions, their approach sheds new light on the complex nature of cooperation and competition in spatially structured environments.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Yanjun Sun , Amanda Q. Nguyen , Joseph P. Nguyen , Luc Le , Dieter Saur , Jiwon Choi , Edward M. Callaway , Xiangmin Xu We developed and applied a Cre-dependent, genetically modified rabies-based tracing system to map direct synaptic connections to specific CA1 neuron types in the mouse hippocampus. We found common inputs to excitatory and inhibitory CA1 neurons from CA3, CA2, the entorhinal cortex (EC), the medial septum (MS), and, unexpectedly, the subiculum. Excitatory CA1 neurons receive inputs from both cholinergic and GABAergic MS neurons, whereas inhibitory neurons receive a great majority of inputs from GABAergic MS neurons. Both cell types also receive weaker input from glutamatergic MS neurons. Comparisons of inputs to CA1 PV+ interneurons versus SOM+ interneurons showed similar strengths of input from the subiculum, but PV+ interneurons received much stronger input than SOM+ neurons from CA3, the EC, and the MS. Thus, rabies tracing identifies hippocampal circuit connections and maps how the different input sources to CA1 are distributed with different strengths on each of its constituent cell types. Graphical abstract Teaser New advances in virology and genetic technology offer powerful tools for mapping cell-type-specific circuit connectivity and function. Sun et al. have developed and applied a new Cre-dependent, genetically modified, rabies-based tracing system to map monosynaptic circuit connections to specific neuron types in hippocampal CA1. This study reveals hippocampal circuit connections and addresses how the different sources of input to CA1 are distributed with different strengths onto each of its constituent cell types.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Alicja M. Ritz , Mark Trautwein , Franziska Grassinger , Anne Spang Prion and prion-like domains (PLDs) are found in many proteins throughout the animal kingdom. We found that the PLD in the S. cerevisiae exomer-dependent cargo protein Pin2 is involved in the regulation of protein transport and localization. The domain serves as a Pin2 retention signal in the trans -Golgi network (TGN). Pin2 is localized in a polarized fashion at the plasma membrane of the bud early in the cell cycle and the bud neck at cytokinesis. This polarized localization is dependent on both exo- and endocytosis. Upon environmental stress, Pin2 is rapidly endocytosed, and the PLD aggregates and causes sequestration of Pin2. The aggregation of Pin2 is reversible upon stress removal and Pin2 is rapidly re-exported to the plasma membrane. Altogether, these data uncover a role for PLDs as protein localization elements. Graphical abstract Teaser Prion-like domains (PLDs) occur in 0.3%–2.5% of cellular proteins, depending on the organism. Despite their relative abundance, the function of PLDs remains unclear. Now, Spang et al. show that a PLD can regulate trafficking/retention of a protein. The Pin2 PLD regulates protein distribution between the trans -Golgi network (TGN) and plasma membrane. Under stress, the PLD causes TGN sequestration of Pin2. Stress release causes rapid export of Pin2 to the plasma membrane.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Natalia Salvadores , Mohammad Shahnawaz , Elio Scarpini , Fabrizio Tagliavini , Claudio Soto Alzheimer's disease (AD) diagnosis is hampered by the lack of early, sensitive, and objective laboratory tests. We describe a sensitive method for biochemical diagnosis of AD based on specific detection of misfolded Aβ oligomers, which play a central role in AD pathogenesis. The protein misfolding cyclic amplification assay (Aβ-PMCA), exploits the functional property of Aβ oligomers to seed the polymerization of monomeric Aβ. Aβ-PMCA allowed detection of as little as 3 fmol of Aβ oligomers. Most importantly, using cerebrospinal fluid, we were able to distinguish AD patients from control individuals affected by a variety of other neurodegenerative disorders or nondegenerative neurological diseases with overall sensitivity of 90% and specificity of 92%. These findings provide the proof-of-principle basis for developing a highly sensitive and specific biochemical test for AD diagnosis. Graphical abstract Teaser Here, Salvadores et al. describe the development of a technology to detect misfolded Aβ oligomers, which are considered the culprits of AD neurodegeneration. The strategy uses the functional property of oligomers to seed the polymerization of monomeric Aβ as a way to measure their presence in biological fluids. Using this approach, the authors could detect Aβ oligomers that were present in femtomolar concentrations. They also detect Aβ oligomers in cerebrospinal fluid from AD patients and can clearly distinguish these samples from control individuals and patients affected by other neurodegenerative diseases.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Chiara Degirolamo , Stefania Rainaldi , Fabiola Bovenga , Stefania Murzilli , Antonio Moschetta Gut microbiota influences host health status by providing trophic, protective, and metabolic functions, including bile acid (BA) biotransformation. Microbial imprinting on BA signature modifies pool size and hydrophobicity, thus contributing to BA enterohepatic circulation. Microbiota-targeted therapies are now emerging as effective strategies for preventing and/or treating gut-related diseases. Here, we show that gut microbiota modulation induced by VSL#3 probiotics enhances BA deconjugation and fecal excretion in mice. These events are associated with changes in ileal BA absorption, repression of the enterohepatic farnesoid X receptor-fibroblast growth factor 15 (FXR-FGF15) axis, and increased hepatic BA neosynthesis. Treatment with a FXR agonist normalized fecal BA levels in probiotic-administered mice, whereas probiotic-induced alterations in BA metabolism are abolished upon FXR and FGF15 deficiency. Our data provide clear in vivo evidence that VSL#3 probiotics promote ileal BA deconjugation with subsequent fecal BA excretion and induce hepatic BA neosynthesis via downregulation of the gut-liver FXR-FGF15 axis. Graphical abstract Teaser A mutual relationship exists between gut microbiota and bile acid (BA) metabolism. Gut microbes influence BA pool size, whereas dysbiosis is associated with abnormalities in BA metabolism. Degirolamo et al. now provide evidence in mice that modifying the microbiota with probiotics leads to significant alteration of the BA profile, including enhanced BA deconjugation and fecal excretion as well as increased hepatic de novo BA synthesis via downregulation of the FXR/Fgf15 gut-liver axis.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Panos Oikonomou , Hani Goodarzi , Saeed Tavazoie Posttranscriptional regulatory programs governing diverse aspects of RNA biology remain largely uncharacterized. Understanding the functional roles of RNA cis -regulatory elements is essential for decoding complex programs that underlie the dynamic regulation of transcript stability, splicing, localization, and translation. Here, we describe a combined experimental/computational technology to reveal a catalog of functional regulatory elements embedded in 3′ UTRs of human transcripts. We used a bidirectional reporter system coupled with flow cytometry and high-throughput sequencing to measure the effect of short, noncoding, vertebrate-conserved RNA sequences on transcript stability and translation. Information-theoretic motif analysis of the resulting sequence-to-gene-expression mapping revealed linear and structural RNA cis- regulatory elements that positively and negatively modulate the posttranscriptional fates of human transcripts. This combined experimental/computational strategy can be used to systematically characterize the vast landscape of posttranscriptional regulatory elements controlling physiological and pathological cellular state transitions. Graphical abstract Teaser Tavazoie and colleagues now report a new experimental/computational framework that allows the systematic and unbiased characterization of conserved 3′ UTR sequences in mammalian cells. They identify a catalog of over 2,000 sequences with strong positive or negative contributions to gene expression. Application of a rigorous information-theoretic approach enables de novo discovery of structural and linear regulatory motifs with significant posttranscriptional regulatory activity.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Haihui Pan , Kunhua Qin , Zhanyong Guo , Yonggang Ma , Craig April , Xiaoli Gao , Thomas G. Andrews , Alex Bokov , Jianhua Zhang , Yidong Chen , Susan T. Weintraub , Jian-Bing Fan , Degeng Wang , Yanfen Hu , Gregory J. Aune , Merry L. Lindsey , Rong Li Negative elongation factor (NELF) is known to enforce promoter-proximal pausing of RNA polymerase II (Pol II), a pervasive phenomenon observed across multicellular genomes. However, the physiological impact of NELF on tissue homeostasis remains unclear. Here, we show that whole-body conditional deletion of the B subunit of NELF ( NELF-B ) in adult mice results in cardiomyopathy and impaired response to cardiac stress. Tissue-specific knockout of NELF-B confirms its cell-autonomous function in cardiomyocytes. NELF directly supports transcription of those genes encoding rate-limiting enzymes in fatty acid oxidation (FAO) and the tricarboxylic acid (TCA) cycle. NELF also shares extensively transcriptional target genes with peroxisome proliferator-activated receptor α (PPARα), a master regulator of energy metabolism in the myocardium. Mechanistically, NELF helps stabilize the transcription initiation complex at the metabolism-related genes. Our findings strongly indicate that NELF is part of the PPARα-mediated transcription regulatory network that maintains metabolic homeostasis in cardiomyocytes. Graphical abstract Teaser Promoter-proximal pausing of RNA polymerase II (Pol II) is a widespread phenomenon in higher eukaryotes. NELF is a critical Pol II-pausing factor in higher multicellular organisms. Pan et al. now uncover a critical role for NELF in cardiac function. Furthermore, their work reveals a previously unrecognized link between NELF and the PPARα-dependent regulatory network that controls elevated energy production in cardiomyocytes.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Zuzana Hořejší , Lasse Stach , Thomas G. Flower , Dhira Joshi , Helen Flynn , J. Mark Skehel , Nicola J. O’Reilly , Roksana W. Ogrodowicz , Stephen J. Smerdon , Simon J. Boulton The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit. Graphical abstract Teaser The R2TP complex is an HSP90 cochaperone that facilitates the assembly of large protein and ribonucleoprotein complexes, but the molecular basis of substrate recognition was not known. Now, Hořejší et al. identify a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex. Structural and proteomic analysis of PIH-N reveals that substrates of the R2TP complex are designated by phosphorylation and recognized by the phosphopeptide binding function of the PIH1D1 subunit.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Mayumi Kitagawa , Suet Yin Sarah Fung , Umar Farook Shahul Hameed , Hidemasa Goto , Masaki Inagaki , Sang Hyun Lee The chromosome passenger complex (CPC) must relocate from anaphase chromosomes to the cell equator for successful cytokinesis. Although this landmark event requires the mitotic kinesin MKlp2, the spatiotemporal mechanistic basis remains elusive. Here, we show that phosphoregulation of MKlp2 by the mitotic kinase Cdk1/cyclin B1 coordinates proper mitotic transition with CPC relocation. We identified multiple Cdk1/cyclin B1 phosphorylation sites within the stalk and C-terminal tail that inhibit microtubule binding and bundling, oligomerization/clustering, and chromosome targeting of MKlp2. Specifically, inhibition of these abilities by Cdk1/cyclin B1 phosphorylation is essential for proper early mitotic progression. Upon anaphase onset, however, reversal of Cdk1/cyclin B1 phosphorylation promotes MKlp2-CPC complex formation and relocates the CPC from anaphase chromosomes for successful cytokinesis. Thus, we propose that phosphoregulation of MKlp2 by Cdk1/cyclin B1 ensures that activation of MKlp2 kinesin and relocation of the CPC occur at the appropriate time and space for proper mitotic progression and genomic stability. Graphical abstract Teaser To orchestrate faithful segregation of sister chromatids to daughter cells with cytokinesis, the chromosome passenger complex (CPC) must relocate from anaphase chromosomes to the cell equator, a process that requires the mitotic kinesin MKlp2. Kitagawa et al. show that phosphoregulation of MKlp2 by the mitotic kinase Cdk1/cyclin B1 coordinates proper mitotic transition with CPC relocation. They provide a mechanistic basis for the spatiotemporal recognition and redistribution of the CPC by the motor kinesin that represents the landmark event of cytokinesis.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Anoushka Davé , Carol Cooley , Mansi Garg , Alessandro Bianchi The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early replication of telomeres in budding yeast and misregulation of origin firing in fission yeast. Several lines of evidence indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase. Our data suggest that the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the mechanism of action of Rif1 in the control of DNA replication and demonstrate a role of PP1 phosphatases in the regulation of origin firing. Graphical abstract Teaser The eukaryotic genome is replicated according to a strict temporal program. Here, Bianchi and colleagues find that Rif1, a master regulator of the DNA replication program in yeast and mammals, exerts its effect on DNA replication origins by recruiting protein phosphatase 1 (PP1) to chromosomes. Rif1/PP1 counteracts the positive action of the DDK kinase on the replicative kinase MCM. In a final twist, kinase action on Rif1 is proposed to eventually release PP1 and allow origin firing.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Hui-Ying Lim , Weidong Wang , Jianming Chen , Karen Ocorr , Rolf Bodmer Reactive oxygen species (ROS) can act cell autonomously and in a paracrine manner by diffusing into nearby cells. Here, we reveal a ROS-mediated paracrine signaling mechanism that does not require entry of ROS into target cells. We found that under physiological conditions, nonmyocytic pericardial cells (PCs) of the Drosophila heart contain elevated levels of ROS compared to the neighboring cardiomyocytes (CMs). We show that ROS in PCs act in a paracrine manner to regulate normal cardiac function, not by diffusing into the CMs to exert their function, but by eliciting a downstream D-MKK3-D-p38 MAPK signaling cascade in PCs that acts on the CMs to regulate their function. We find that ROS-D-p38 signaling in PCs during development is also important for establishing normal adult cardiac function. Our results provide evidence for a previously unrecognized role of ROS in mediating PC/CM interactions that significantly modulates heart function. Graphical abstract Teaser Reactive oxygen species (ROS) can act autonomously within a tissue or signal in a paracrine fashion by diffusion. Here, Lim et al. find that altering physiological ROS level in nonmyocytic pericardial cells of the Drosophila heart affects the function of neighboring cardiomyocytes. However, ROS do not seem to act by diffusion into the myocardium but rather by activating a p38 signaling cascade within pericardial cells that then regulates cardiac function, possibly by modulating cell-cell contact.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Abbas Hadji , Paolo Ceppi , Andrea E. Murmann , Sonia Brockway , Abhinandan Pattanayak , Bhavneet Bhinder , Annika Hau , Shirley De Chant , Vamsi Parimi , Piotre Kolesza , JoAnne Richards , Navdeep Chandel , Hakim Djaballah , Marcus E. Peter CD95 (Fas/APO-1), when bound by its cognate ligand CD95L, induces cells to die by apoptosis. We now show that elimination of CD95 or CD95L results in a form of cell death that is independent of caspase-8, RIPK1/MLKL, and p53, is not inhibited by Bcl-x L expression, and preferentially affects cancer cells. All tumors that formed in mouse models of low-grade serous ovarian cancer or chemically induced liver cancer with tissue-specific deletion of CD95 still expressed CD95, suggesting that cancer cannot form in the absence of CD95. Death induced by CD95R/L elimination (DICE) is characterized by an increase in cell size, production of mitochondrial ROS, and DNA damage. It resembles a necrotic form of mitotic catastrophe. No single drug was found to completely block this form of cell death, and it could also not be blocked by the knockdown of a single gene, making it a promising way to kill cancer cells. Graphical abstract Teaser CD95 is a well-studied receptor that mediates cell death (apoptosis) when stimulated by its cognate ligand, CD95L. Hadji et al. report that when either CD95 or CD95L is removed, cancer cells in vitro and in vivo undergo a vigorous form of cell death called DICE. DICE represents multiple different cell death pathways and cannot be blocked by a number of drugs. It does not depend on any single gene, suggesting that DICE could be used to kill cancer cells.

Description: Publication date: Available online 20 March 2014 Source: Cell Reports Author(s): Cristina Cebrian , Naoya Asai , Vivette D’Agati , Frank Costantini Nephrons, the functional units of the kidney, develop from progenitor cells (cap mesenchyme [CM]) surrounding the epithelial ureteric bud (UB) tips. Reciprocal signaling between UB and CM induces nephrogenesis and UB branching. Although low nephron number is implicated in hypertension and renal disease, the mechanisms that determine nephron number are obscure. To test the importance of nephron progenitor cell number, we genetically ablated 40% of these cells, asking whether this would limit kidney size and nephron number or whether compensatory mechanisms would allow the developing organ to recover. The reduction in CM cell number decreased the rate of branching, which in turn allowed the number of CM cells per UB tip to normalize, revealing a self-correction mechanism. However, the retarded UB branching impaired kidney growth, leaving a permanent nephron deficit. Thus, the number of fetal nephron progenitor cells is an important determinant of nephron endowment, largely via its effect on UB branching. Graphical abstract Teaser The number of nephrons (the filtering units of the kidney) varies widely among individuals, and a nephron deficit may cause hypertension and chronic kidney disease. To investigate the developmental mechanisms that control nephron number, Costantini et al. genetically ablated a fraction of nephron-forming cells in fetal mice to ask whether compensatory mechanisms would restore nephron number during fetal or postnatal development. Instead, a failure of compensation revealed that the number of fetal progenitor cells limits final nephron endowment in adult kidneys.

Description: Publication date: Available online 23 January 2014 Source: Cell Reports Author(s): Brad J. Niles , Amelia C. Joslin , Tara Fresques , Ted Powers Reactive oxygen species (ROS) are produced during normal metabolism and can function as signaling molecules. However, ROS at elevated levels can damage cells. Here, we identify the conserved target of rapamycin complex 2 (TORC2)/Ypk1 signaling module as an important regulator of ROS in the model eukaryotic organism, S. cerevisiae . We show that TORC2/Ypk1 suppresses ROS produced both by mitochondria as well as by nonmitochondrial sources, including changes in acidification of the vacuole. Furthermore, we link vacuole-related ROS to sphingolipids, essential components of cellular membranes, whose synthesis is also controlled by TORC2/Ypk1 signaling. In total, our data reveal that TORC2/Ypk1 act within a homeostatic feedback loop to maintain sphingolipid levels and that ROS are a critical regulatory signal within this system. Thus, ROS sensing and signaling by TORC2/Ypk1 play a central physiological role in sphingolipid biosynthesis and in the maintenance of cell growth and viability. Graphical abstract Teaser Although reactive oxygen species (ROS) are products of normal metabolism, elevated ROS can damage cells. In this study, Powers and colleagues identify the TORC2 branch of the highly conserved TOR signaling network as an important regulator of ROS in S. cerevisiae . The data reveal that TORC2 acts through its downstream kinase Ypk1 to regulate ROS from multiple sources. In addition, ROS participates in a feedback loop to maintain sphingolipid homeostasis by promoting increased TORC2 phosphorylation of Ypk1.

Description: Publication date: Available online 23 January 2014 Source: Cell Reports Author(s): Cédric Delevoye , Stéphanie Miserey-Lenkei , Guillaume Montagnac , Floriane Gilles-Marsens , Perrine Paul-Gilloteaux , Francesca Giordano , François Waharte , Michael S. Marks , Bruno Goud , Graça Raposo Early endosomes consist of vacuolar sorting and tubular recycling domains that segregate components fated for degradation in lysosomes or reuse by recycling to the plasma membrane or Golgi. The tubular transport intermediates that constitute recycling endosomes function in cell polarity, migration, and cytokinesis. Endosomal tubulation and fission require both actin and intact microtubules, but although factors that stabilize recycling endosomal tubules have been identified, those required for tubule generation from vacuolar sorting endosomes (SEs) remain unknown. We show that the microtubule motor KIF13A associates with recycling endosome tubules and controls their morphogenesis. Interfering with KIF13A function impairs the formation of endosomal tubules from SEs with consequent defects in endosome homeostasis and cargo recycling. Moreover, KIF13A interacts and cooperates with RAB11 to generate endosomal tubules. Our data illustrate how a microtubule motor couples early endosome morphogenesis to its motility and function. Graphical abstract Teaser Recycling endosomes are tubules that originate from vacuolar early endosomes. In this study, Delevoye and colleagues identify the kinesin-3 KIF13A as a key component for endocytic tubule morphogenesis. Impairing KIF13A function affects endosomal tubule formation and cargo recycling back to the plasma membrane. Moreover, KIF13A interacts and cooperates with the small GTPase RAB11 in order to generate tubular recycling intermediates. These data highlight how a microtubule-based motor coordinates early endosome biogenesis, motility, and function.

Description: Publication date: Available online 23 January 2014 Source: Cell Reports Author(s): Song Eun Lee , Aiko Sada , Meng Zhang , David J. McDermitt , Shu Yang Lu , Kenneth J. Kemphues , Tudorita Tumbar Quiescent hair follicle (HF) bulge stem cells (SCs) differentiate to early progenitor (EP) hair germ (HG) cells, which divide to produce transit-amplifying matrix cells. EPs can revert to SCs upon injury, but whether this dedifferentiation occurs in normal HF homeostasis (hair cycle) and the mechanisms regulating both differentiation and dedifferentiation are unclear. Here, we use lineage tracing, gain of function, transcriptional profiling, and functional assays to examine the role of observed endogenous Runx1 level changes in the hair cycle. We find that forced Runx1 expression induces hair degeneration (catagen) and simultaneously promotes changes in the quiescent bulge SC transcriptome toward a cell state resembling the EP HG fate. This cell-state transition is functionally reversible. We propose that SC differentiation and dedifferentiation are likely to occur during normal HF degeneration and niche restructuring in response to changes in endogenous Runx1 levels associated with SC location with respect to the niche. Graphical abstract Teaser In this study, Tumbar and colleagues show that the transcription factor Runx1 induces reversible differentiation (dedifferentiation) of quiescent hair follicle stem cells to early progenitors. Based on lineage tracing, target gene analysis, and gain of function studies, the authors suggest that dedifferentiation occurs normally in somatic stem cells to confer robustness in adult-tissue homeostasis. Reversible cell-fate transitions in mammals were previously described in the germ line, in oncogenic transformation, or after injury induced by targeted cell ablation.

Description: Publication date: Available online 30 January 2014 Source: Cell Reports Author(s): Rémi Buisson , Joshi Niraj , Joris Pauty , Ranjan Maity , Weixing Zhao , Yan Coulombe , Patrick Sung , Jean-Yves Masson One envisioned function of homologous recombination (HR) is to find a template for DNA synthesis from the resected 3′-OH molecules that occur during double-strand break (DSB) repair at collapsed replication forks. However, the interplay between DNA synthesis and HR remains poorly understood in higher eukaryotic cells. Here, we reveal functions for the breast cancer proteins BRCA2 and PALB2 at blocked replication forks and show a role for these proteins in stimulating polymerase η (Polη) to initiate DNA synthesis. PALB2, BRCA2, and Polη colocalize at stalled or collapsed replication forks after hydroxyurea treatment. Moreover, PALB2 and BRCA2 interact with Polη and are required to sustain the recruitment of Polη at blocked replication forks. PALB2 and BRCA2 stimulate Polη-dependent DNA synthesis on D loop substrates. We conclude that PALB2 and BRCA2, in addition to their functions in D loop formation, play crucial roles in the initiation of recombination-associated DNA synthesis by Polη-mediated DNA repair. Graphical abstract Teaser BRCA2 and PALB2 are two important regulators for the repair of DNA double-strand breaks. Because of their large protein size and multiple domains, many functions remain to be uncovered for these proteins. Here, Masson and colleagues advance our understanding of BRCA2 and PALB2 functions. In addition to their function in regulating the RAD51 recombinase, PALB2 and BRCA2 stimulate DNA synthesis by polymerase η, facilitating DNA repair upon DNA replication stress.

Description: Publication date: Available online 27 February 2014 Source: Cell Reports Author(s): Dominique Cannella , Marie-Pierre Brenier-Pinchart , Laurence Braun , Jason M. van Rooyen , Alexandre Bougdour , Olivier Bastien , Michael S. Behnke , Rose-Laurence Curt , Aurélie Curt , Jeroen P.J. Saeij , L. David Sibley , Hervé Pelloux , Mohamed-Ali Hakimi microRNAs were recently found to be regulators of the host response to infection by apicomplexan parasites. In this study, we identified two immunomodulatory microRNAs, miR-146a and miR-155, that were coinduced in the brains of mice challenged with Toxoplasma in a strain-specific manner. These microRNAs define a characteristic fingerprint for infection by type II strains, which are the most prevalent cause of human toxoplasmosis in Europe and North America. Using forward genetics, we showed that strain-specific differences in miR-146a modulation were in part mediated by the rhoptry kinase, ROP16. Remarkably, we found that miR-146a deficiency led to better control of parasite burden in the gut and most likely of early parasite dissemination in the brain tissue, resulting in the long-term survival of mice. Graphical abstract Teaser The obligate intracellular parasite Toxoplasma gondii has developed tactics to interfere with the host miRNA profile in a strain-specific manner. In this study, Hakimi and colleagues uncover a characteristic microRNA fingerprint (miR-146a/miR-155) that typifies Toxoplasma long-term persistence and latency in the brain of mice chronically infected by type II strains, which are the most prevalent cause of human toxoplasmosis in Europe and North America.

Description: Publication date: Available online 27 February 2014 Source: Cell Reports Author(s): Tadahiro Nagaoka , Riuko Ohashi , Ayumu Inutsuka , Seiko Sakai , Nobuyoshi Fujisawa , Minesuke Yokoyama , Yina H. Huang , Michihiro Igarashi , Masashi Kishi Although regulators of the Wnt/planar cell polarity (PCP) pathway are widely expressed in vertebrate nervous systems, their roles at synapses are unknown. Here, we show that Vangl2 is a postsynaptic factor crucial for synaptogenesis and that it coprecipitates with N-cadherin and PSD-95 from synapse-rich brain extracts. Vangl2 directly binds N-cadherin and enhances its internalization in a Rab5-dependent manner. This physical and functional interaction is suppressed by β-catenin, which binds the same intracellular region of N-cadherin as Vangl2. In hippocampal neurons expressing reduced Vangl2 levels, dendritic spine formation as well as synaptic marker clustering is significantly impaired. Furthermore, Prickle2, another postsynaptic PCP component, inhibits the N-cadherin-Vangl2 interaction and is required for normal spine formation. These results demonstrate direct control of classic cadherin by PCP factors; this control may play a central role in the precise formation and maturation of cell-cell adhesions at the synapse. Graphical abstract Teaser Planar cell polarity (PCP) signaling regulates tissue morphogenesis, including the uniform orientation of cochlear hair cells, convergent extension movements of the lateral mesoderm, and ommatidial rotation in the Drosophila eye. In this study, Kishi and colleagues report a role for PCP regulators in animal development. Vangl2 and Prickle2—components of the Wnt/PCP pathway—are required for the normal development of CNS synapses. The underlying mechanism involves the direct control of postsynaptic N-cadherin transport by the PCP regulators.

Description: Publication date: Available online 13 February 2014 Source: Cell Reports Author(s): Laurence Decourty , Antonia Doyen , Christophe Malabat , Emmanuel Frachon , Delphine Rispal , Bertrand Séraphin , Frank Feuerbach , Alain Jacquier , Cosmin Saveanu Nonsense-mediated mRNA decay (NMD) destabilizes eukaryotic transcripts with long 3′ UTRs. To investigate whether other transcript features affect NMD, we generated yeast strains expressing chromosomal-derived mRNAs with 979 different promoter and open reading frame (ORF) regions and with the same long, destabilizing 3′ UTR. We developed a barcode-based DNA microarray strategy to compare the levels of each reporter mRNA in strains with or without active NMD. The size of the coding region had a significant negative effect on NMD efficiency. This effect was not specific to the tested 3′ UTR because two other different NMD reporters became less sensitive to NMD when ORF length was increased. Inefficient NMD was not due to a lack of association of Upf1 to long ORF transcripts. In conclusion, in addition to a long 3′ UTR, short translation length is an important feature of NMD substrates in yeast. Graphical abstract Teaser Nonsense-mediated mRNA decay (NMD) is the most studied pathway for degradation of “aberrant” mRNA. In this study, Jacquier, Saveanu, and colleagues show that molecular barcodes can be used to perform large-scale studies of RNA reporters in the yeast S. cerevisiae . Testing hundreds of NMD reporters revealed a strong correlation between coding sequence size and transcript stability in an NMD context. These data suggest hypotheses about how NMD substrates are detected and degraded in eukaryotes.

Description: Publication date: Available online 13 February 2014 Source: Cell Reports Author(s): Catharina Arnold-Schrauf , Markus Dudek , Anastasia Dielmann , Luigia Pace , Maxine Swallow , Friederike Kruse , Anja A. Kühl , Bernhard Holzmann , Luciana Berod , Tim Sparwasser Listeria monocytogenes (LM), a facultative intracellular Gram-positive pathogen, can cause life-threatening infections in humans. In mice, the signaling cascade downstream of the myeloid differentiation factor 88 (MyD88) is essential for proper innate immune activation against LM, as MyD88-deficient mice succumb early to infection. Here, we show that MyD88 signaling in dendritic cells (DCs) is sufficient to mediate the protective innate response, including the production of proinflammatory cytokines, neutrophil infiltration, bacterial clearance, and full protection from lethal infection. We also demonstrate that MyD88 signaling by DCs controls the infection rates of CD8α + cDCs and thus limits the spread of LM to the T cell areas. Furthermore, in mice expressing MyD88 in DCs, inflammatory monocytes, which are required for bacterial clearance, are activated independently of intrinsic MyD88 signaling. In conclusion, CD11c + conventional DCs critically integrate pathogen-derived signals via MyD88 signaling during early infection with LM in vivo. Graphical abstract Teaser Innate immune activation via the MyD88 signaling pathway is a key event during many infections. However, whether cell-type-specific signaling in dendritic cells (DCs) is sufficient for infection control is unknown. Here, Sparwasser and colleagues demonstrate that MyD88 signaling in DCs critically coordinates innate immune activation and thereby prevents bacterial dissemination during infection with Listeria monocytogenes , whereas its function in other myeloid cells, such as inflammatory monocytes, is dispensable. These findings suggest that MyD88-dependent DC activation initiates bacterial infection control.

Description: Publication date: Available online 13 February 2014 Source: Cell Reports Author(s): Régis Stentz , Samantha Osborne , Nikki Horn , Arthur W.H. Li , Isabelle Hautefort , Roy Bongaerts , Marine Rouyer , Paul Bailey , Stephen B. Shears , Andrew M. Hemmings , Charles A. Brearley , Simon R. Carding Dietary InsP 6 can modulate eukaryotic cell proliferation and has complex nutritive consequences, but its metabolism in the mammalian gastrointestinal tract is poorly understood. Therefore, we performed phylogenetic analyses of the gastrointestinal microbiome in order to search for candidate InsP 6 phosphatases. We determined that prominent gut bacteria express homologs of the mammalian InsP 6 phosphatase (MINPP) and characterized the enzyme from Bacteroides thetaiotaomicron (BtMinpp). We show that BtMinpp has exceptionally high catalytic activity, which we rationalize on the basis of mutagenesis studies and by determining its crystal structure at 1.9 Å resolution. We demonstrate that BtMinpp is packaged inside outer membrane vesicles (OMVs) protecting the enzyme from degradation by gastrointestinal proteases. Moreover, we uncover an example of cross-kingdom cell-to-cell signaling, showing that the BtMinpp-OMVs interact with intestinal epithelial cells to promote intracellular Ca 2+ signaling. Our characterization of BtMinpp offers several directions for understanding how the microbiome serves human gastrointestinal physiology. Graphical abstract Teaser Brearley, Carding, and colleagues now find that the prevalent gut bacterium Bacteroides thetaiotaomicron (Bt) contributes to human physiology, metabolism, and homeostasis through gastrointestinal InsP 6 metabolism and secretion of a mammalian cell-signaling InsP 6 phosphatase, MINPP. BtMinpp catalytic activity was exceptionally high and rationalized from its crystal structure. BtMinpp is packaged inside outermembrane vesicles (OMVs) protecting against intestinal proteases. BtMinpp and OMVs also promoted Ca 2+ signaling in intestinal epithelial cells, an example of cross-kingdom dialog.

Description: Publication date: Available online 27 February 2014 Source: Cell Reports Author(s): Federica Parisi , Rhoda K. Stefanatos , Karen Strathdee , Yachuan Yu , Marcos Vidal High tumor burden is associated with increased levels of circulating inflammatory cytokines that influence the pathophysiology of the tumor and its environment. The cellular and molecular events mediating the organismal response to a growing tumor are poorly understood. Here, we report a bidirectional crosstalk between epithelial tumors and the fat body—a peripheral immune tissue—in Drosophila . Tumors trigger a systemic immune response through activation of Eiger/TNF signaling, which leads to Toll pathway upregulation in adipocytes. Reciprocally, Toll elicits a non-tissue-autonomous program in adipocytes, which drives tumor cell death. Hemocytes play a critical role in this system by producing the ligands Spätzle and Eiger, which are required for Toll activation in the fat body and tumor cell death. Altogether, our results provide a paradigm for a long-range tumor suppression function of adipocytes in Drosophila , which may represent an evolutionarily conserved mechanism in the organismal response to solid tumors. Graphical abstract Teaser In this study, Vidal and colleagues show that, in a Drosophila model of cancer, epithelial tumors trigger long-range activation of Toll signaling in adipocytes. In turn, Toll pathway activation in adipocytes stimulates apoptosis in the epithelial tumors. This crosstalk between epithelial tumors and the fat body is mediated by the macrophage-like hemocytes, via the production of the ligands Spätzle and Eiger, which respectively trigger the Toll pathway in adipocytes and the JNK pathway in tumors.

Description: Publication date: Available online 27 February 2014 Source: Cell Reports Author(s): Andrey S. Selyunin , Lovett Evan Reddick , Bethany A. Weigele , Neal M. Alto Bidirectional vesicular transport between the endoplasmic reticulum (ER) and Golgi is mediated largely by ARF and Rab GTPases, which orchestrate vesicle fission and fusion, respectively. How their activities are coordinated in order to define the successive steps of the secretory pathway and preserve traffic directionality is not well understood in part due to the scarcity of molecular tools that simultaneously target ARF and Rab signaling. Here, we take advantage of the unique scaffolding properties of E. coli secreted protein G (EspG) to describe the critical role of ARF1/Rab1 spatiotemporal coordination in vesicular transport at the ER-Golgi intermediate compartment. Structural modeling and cellular studies show that EspG induces bidirectional traffic arrest by tethering vesicles through select ARF1-GTP/effector complexes and local inactivation of Rab1. The mechanistic insights presented here establish the effectiveness of a small bacterial catalytic scaffold for studying complex processes and reveal an alternative mechanism of immune regulation by an important human pathogen. Graphical abstract Teaser Endomembrane trafficking is a dynamic process that is essential for cellular homeostasis, signal transduction, and immunity, but it has been difficult to elucidate signaling crosstalk involved in the regulation of its individual stages. Here, Alto and colleagues describe a unique molecular mechanism of a bacterial effector protein EspG that uses scaffolding properties to inhibit host protein secretion. Through simultaneous coordination of ARF1 and Rab1 GTPase activities, EspG selectively promotes the tethering of membranes via GMAP-210, which leads to bidirectional vesicular traffic arrest and disassembly of the Golgi apparatus.

Description: Publication date: Available online 27 February 2014 Source: Cell Reports Author(s): Gray R. Lyons , Ryan O. Andersen , Khadar Abdi , Won-Seok Song , Chay T. Kuo Dendrites often exhibit structural changes in response to local inputs. Although mechanisms that pattern and maintain dendritic arbors are becoming clearer, processes regulating regrowth, during context-dependent plasticity or after injury, remain poorly understood. We found that a class of Drosophila sensory neurons, through complete pruning and regeneration, can elaborate two distinct dendritic trees, innervating independent sensory fields. An expression screen identified Cysteine proteinase-1 ( Cp1 ) as a critical regulator of this process. Unlike known ecdysone effectors, Cp1 -mutant ddaC neurons pruned larval dendrites normally but failed to regrow adult dendrites. Cp1 expression was upregulated/concentrated in the nucleus during metamorphosis, controlling production of a truncated Cut homeodomain transcription factor. This truncated Cut, but not the full-length protein, allowed Cp1 -mutant ddaC neurons to regenerate higher-order adult dendrites. These results identify a molecular pathway needed for dendrite regrowth after pruning, which allows the same neuron to innervate distinct sensory fields. Graphical abstract Teaser Dendrites are the primary antennas for information input to neurons. Their territory for individual neurons represents a "receptive or sensory field" and is important for conveying the precision of information gathered. But when this sensory field changes due to experience or injury, how does a neuron innervate a new sensory field? Does it reuse a developmental program first employed to grow dendrites, or does it call up a new one? In this study, Kuo and colleagues show that, surprisingly, it is both.

Description: Publication date: 27 March 2014 Source: Cell Reports, Volume 6, Issue 6 Author(s): Caleb McKinney , Jiri Zavadil , Christopher Bianco , Lora Shiflett , Stuart Brown , Ian Mohr

Description: Publication date: 27 March 2014 Source: Cell Reports, Volume 6, Issue 6 Author(s): Dan Xu , Feng Zhang , Yaqing Wang , Yiming Sun , Zhiheng Xu

Description: Publication date: 27 March 2014 Source: Cell Reports, Volume 6, Issue 6 Author(s): Andrew R. Bassett , Charlotte Tibbit , Chris P. Ponting , Ji-Long Liu

Description: Publication date: Available online 27 March 2014 Source: Cell Reports Author(s): Emanuela Zuccaro , Matteo Bergami , Beatrice Vignoli , Guillaume Bony , Brian A. Pierchala , Spartaco Santi , Laura Cancedda , Marco Canossa Newly generated neurons initiate polarizing signals that specify a single axon and multiple dendrites, a process critical for patterning neuronal circuits in vivo. Here, we report that the pan-neurotrophin receptor p75 NTR is a polarity regulator that localizes asymmetrically in differentiating neurons in response to neurotrophins and is required for specification of the future axon. In cultured hippocampal neurons, local exposure to neurotrophins causes early accumulation of p75 NTR into one undifferentiated neurite to specify axon fate. Moreover, knockout or knockdown of p75 NTR results in failure to initiate an axon in newborn neurons upon cell-cycle exit in vitro and in the developing cortex, as well as during adult hippocampal neurogenesis in vivo. Hence, p75 NTR governs neuronal polarity, determining pattern and assembly of neuronal circuits in adult hippocampus and cortical development. Video Abstract Graphical abstract Teaser Specification of an axon and multiple dendrites determines how a newly generated neuron integrates into a neuronal circuit. In this study, Zuccaro et al. demonstrate a direct link between neurotrophins and pan-neurotrophin receptor p75NTR during axon specification. Notably, they show that p75NTR is required to initiate axons both in vitro and in vivo, yielding fundamental insights into how newly generated neurons integrate into initial circuits of the developing cortex or preexisting circuits in neurogenic regions of the adult hippocampus.