Dynamin-Related Protein 1-Dependent Mitochondrial Fission Changes in the Dorsal Vagal Complex Regulate Insulin Action

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Beatrice M. Filippi, Mona A. Abraham, Pamuditha N. Silva, Mozhgan Rasti, Mary P. LaPierre, Paige V. Bauer, Jonathan V. Rocheleau, Tony K.T. Lam
Mitochondria undergo dynamic changes to maintain function in eukaryotic cells. Insulin action in parallel regulates glucose homeostasis, but whether specific changes in mitochondrial dynamics alter insulin action and glucose homeostasis remains elusive. Here, we report that high-fat feeding in rodents incurred adaptive dynamic changes in mitochondria through an increase in mitochondrial fission in parallel to an activation of dynamin-related protein 1 (Drp1) in the dorsal vagal complex (DVC) of the brain. Direct inhibition of Drp1 negated high-fat-feeding-induced mitochondrial fission, endoplasmic reticulum (ER) stress, and insulin resistance in the DVC and subsequently restored hepatic glucose production regulation. Conversely, molecular activation of DVC Drp1 in healthy rodents was sufficient to induce DVC mitochondrial fission, ER stress, and insulin resistance. Together, these data illustrate that Drp1-dependent mitochondrial fission changes in the DVC regulate insulin action and suggest that targeting the Drp1-mitochondrial-dependent pathway in the brain may have therapeutic potential in insulin resistance.

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Teaser

Filippi et al. find that induction of Drp1-dependent mitochondrial fission in the dorsal vagal complex of rodents under high-fat feeding can induce ER stress and insulin resistance.


http://ift.tt/2lVeyQu

Mutations that Allow SIR2 Orthologs to Function in a NAD+-Depleted Environment

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Caitlin R. Ondracek, Vincent Frappier, Alison E. Ringel, Cynthia Wolberger, Leonard Guarente
Sirtuin enzymes depend on NAD⁺ to catalyze protein deacetylation. Therefore, the lowering of NAD⁺ during aging leads to decreased sirtuin activity and may speed up aging processes in laboratory animals and humans. In this study, we used a genetic screen to identify two mutations in the catalytic domain of yeast Sir2 that allow the enzyme to function in an NAD⁺-depleted environment. These mutant enzymes give rise to a significant increase of yeast replicative lifespan and increase deacetylation by the Sir2 ortholog, SIRT1, in mammalian cells. Our data suggest that these mutations increase the stability of the conserved catalytic sirtuin domain, thereby increasing the catalytic efficiency of the mutant enzymes. Our approach to identifying sirtuin mutants that permit function in NAD⁺-limited environments may inform the design of small molecules that can maintain sirtuin activity in aging organisms.

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Teaser

Ondracek et al. report the identification of two adaptive mutations in yeast Sir2 that allow the enzyme to function in an NAD⁺-depleted environment by enhancing protein stability and increasing enzyme catalysis, which increases yeast replicative lifespan and deacetylation of in vivo substrates by the Sir2 ortholog, SIRT1, in mammalian cells.


http://ift.tt/2lV3rHj

R-spondin1 Controls Muscle Cell Fusion through Dual Regulation of Antagonistic Wnt Signaling Pathways

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Floriane Lacour, Elsa Vezin, C. Florian Bentzinger, Marie-Claude Sincennes, Lorenzo Giordani, Arnaud Ferry, Robert Mitchell, Ketan Patel, Michael A. Rudnicki, Marie-Christine Chaboissier, Anne-Amandine Chassot, Fabien Le Grand
Wnt-mediated signals are involved in many important steps in mammalian regeneration. In multiple cell types, the R-spondin (Rspo) family of secreted proteins potently activates the canonical Wnt/β-catenin pathway. Here, we identify Rspo1 as a mediator of skeletal muscle tissue repair. First, we show that deletion of Rspo1 results in global alteration of muscle regeneration kinetics following acute injury. We find that muscle progenitor cells lacking Rspo1 show delayed differentiation due to reduced activation of Wnt/β-catenin target genes. Furthermore, muscle cells lacking Rspo1 have a fusion phenotype leading to larger myotubes containing supernumerary nuclei both in vitro and in vivo. The increase in muscle fusion was dependent on downregulation of Wnt/β-catenin and upregulation of non-canonical Wnt7a/Fzd7/Rac1 signaling. We conclude that reciprocal control of antagonistic Wnt signaling pathways by Rspo1 in muscle stem cell progeny is a key step ensuring normal tissue architecture restoration following acute damage.

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Teaser

Using mice lacking the Wnt enhancer Rspo1, Lacour et al. demonstrate that Rspo1 controls muscle cell fusion during skeletal muscle regeneration. Rspo1-null muscle progenitor cells differentiate less efficiently but fuse extensively compared to wild-type cells. Rspo1 is shown to regulate the antagonism between canonical and non-canonical Wnt signaling pathways.


http://ift.tt/2lVpwoZ

ATR Mutations Promote the Growth of Melanoma Tumors by Modulating the Immune Microenvironment

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Chi-Fen Chen, Rolando Ruiz-Vega, Priya Vasudeva, Francisco Espitia, Tatiana B. Krasieva, Sebastien de Feraudy, Bruce J. Tromberg, Sharon Huang, Chad P. Garner, Jie Wu, Dave S. Hoon, Anand K. Ganesan
Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet somehow suppress the immune response to facilitate continued growth. In this study, we identify a subset of human melanomas that have loss-of-function mutations in ATR, a kinase that recognizes and repairs UV-induced DNA damage and is required for cellular proliferation. ATR mutant tumors exhibit both the accumulation of multiple mutations and the altered expression of inflammatory genes, resulting in decreased T cell recruitment and increased recruitment of macrophages known to spur tumor invasion. Taken together, these studies identify a mechanism by which melanoma cells modulate the immune microenvironment to promote continued growth.

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Teaser

Tumors grow not only because they acquire mutations that promote their growth but also because they prevent the immune system from recognizing them. Chen et al. identify ATR mutations in human melanoma tumors and determine that ATR mutant melanomas harness the immune system to accelerate tumor growth.


http://ift.tt/2lVe3pC

Interrogation of Functional Cell-Surface Markers Identifies CD151 Dependency in High-Grade Serous Ovarian Cancer

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Mauricio Medrano, Laudine Communal, Kevin R. Brown, Marcin Iwanicki, Josee Normand, Joshua Paterson, Fabrice Sircoulomb, Paul Krzyzanowski, Marian Novak, Sasha A. Doodnauth, Fernando Suarez Saiz, Jane Cullis, Rima Al-awar, Benjamin G. Neel, John McPherson, Ronny Drapkin, Laurie Ailles, Anne-Marie Mes-Massons, Robert Rottapel
The degree of genetic aberrations characteristic of high-grade serous ovarian cancer (HGSC) makes identification of the molecular features that drive tumor progression difficult. Here, we perform genome-wide RNAi screens and comprehensive expression analysis of cell-surface markers in a panel of HGSC cell lines to identify genes that are critical to their survival. We report that the tetraspanin CD151 contributes to survival of a subset of HGSC cell lines associated with a ZEB transcriptional program and supports the growth of HGSC tumors. Moreover, we show that high CD151 expression is prognostic of poor clinical outcome. This study reveals cell-surface vulnerabilities associated with HGSC, provides a framework for identifying therapeutic targets, and reports a role for CD151 in HGSC.

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Teaser

Medrano et al. conduct whole-genome short hairpin RNA screens in 27 high-grade serous ovarian carcinoma (HGSC) cell lines to identify vulnerabilities in HGSC. Analysis of the cell surface reveals that CD151 is essential for cell survival through a ZEB-dependent mechanism.


http://ift.tt/2lV4uqQ

Tissue-Specific Mitochondrial Decoding of Cytoplasmic Ca2+ Signals Is Controlled by the Stoichiometry of MICU1/2 and MCU

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Melanie Paillard, György Csordás, Gergö Szanda, Tünde Golenár, Valentina Debattisti, Adam Bartok, Nadan Wang, Cynthia Moffat, Erin L. Seifert, András Spät, György Hajnóczky
Mitochondrial Ca²⁺ uptake through the Ca²⁺ uniporter supports cell functions, including oxidative metabolism, while meeting tissue-specific calcium signaling patterns and energy needs. The molecular mechanisms underlying tissue-specific control of the uniporter are unknown. Here, we investigated a possible role for tissue-specific stoichiometry between the Ca²⁺-sensing regulators (MICUs) and pore unit (MCU) of the uniporter. Low MICU1:MCU protein ratio lowered the [Ca²⁺] threshold for Ca²⁺ uptake and activation of oxidative metabolism but decreased the cooperativity of uniporter activation in heart and skeletal muscle compared to liver. In MICU1-overexpressing cells, MICU1 was pulled down by MCU proportionally to MICU1 overexpression, suggesting that MICU1:MCU protein ratio directly reflected their association. Overexpressing MICU1 in the heart increased MICU1:MCU ratio, leading to liver-like mitochondrial Ca²⁺ uptake phenotype and cardiac contractile dysfunction. Thus, the proportion of MICU1-free and MICU1-associated MCU controls these tissue-specific uniporter phenotypes and downstream Ca²⁺ tuning of oxidative metabolism.

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Teaser

Paillard et al. report that the relative abundance of the pore-forming protein of the mitochondrial Ca²⁺ uniporter (MCU) and its Ca²⁺-sensing regulator (MICU1) define the proportion of MCU complexes with or without MICU1. This ratio is central to programming tissue-specific mitochondrial Ca²⁺ uptake phenotypes in the heart and liver.


http://ift.tt/2lV82cq

Enhancer-Mediated Oncogenic Function of the Menin Tumor Suppressor in Breast Cancer

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Koen M.A. Dreijerink, Anna C. Groner, Erica S.M. Vos, Alba Font-Tello, Lei Gu, David Chi, Jaime Reyes, Jennifer Cook, Elgene Lim, Charles Y. Lin, Wouter de Laat, Prakash K. Rao, Henry W. Long, Myles Brown
While the multiple endocrine neoplasia type 1 (MEN1) gene functions as a tumor suppressor in a variety of cancer types, we explored its oncogenic role in breast tumorigenesis. The MEN1 gene product menin is involved in H3K4 trimethylation and co-activates transcription. We integrated ChIP-seq and RNA-seq data to identify menin target genes. Our analysis revealed that menin-dependent target gene promoters display looping to distal enhancers that are bound by menin, FOXA1 and GATA3. In this fashion, MEN1 co-regulates a proliferative breast cancer-specific gene expression program in ER⁺ cells. In primary mammary cells, MEN1 exerts an anti-proliferative function by regulating a distinct expression signature. Our findings clarify the cell-type-specific functions of MEN1 and inform the development of menin-directed treatments for breast cancer.

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Teaser

Dreijerink et al. describe the oncogenic actions of the tumor suppressor menin in breast cancer cells. In mammary progenitor cells, menin regulates anti-proliferative genes. Menin is present at FOXA1 and GATA3-bound enhancers that associate with promoters through chromatin looping. Insight into menin's context-dependent function suggests therapeutic strategies.


http://ift.tt/2lV3piF

Circulating NOD1 Activators and Hematopoietic NOD1 Contribute to Metabolic Inflammation and Insulin Resistance

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Kenny L. Chan, Theresa H. Tam, Parastoo Boroumand, David Prescott, Sheila R. Costford, Nichole K. Escalante, Noah Fine, YuShan Tu, Susan J. Robertson, Dilshaayee Prabaharan, Zhi Liu, Philip J. Bilan, Michael W. Salter, Michael Glogauer, Stephen E. Girardin, Dana J. Philpott, Amira Klip
Insulin resistance is a chronic inflammatory condition accompanying obesity or high fat diets that leads to type 2 diabetes. It is hypothesized that lipids and gut bacterial compounds in particular contribute to metabolic inflammation by activating the immune system; however, the receptors detecting these "instigators" of inflammation remain largely undefined. Here, we show that circulating activators of NOD1, a receptor for bacterial peptidoglycan, increase with high fat feeding in mice, suggesting that NOD1 could be a critical sensor leading to metabolic inflammation. Hematopoietic depletion of NOD1 did not prevent weight gain but protected chimeric mice against diet-induced glucose and insulin intolerance. Mechanistically, while macrophage infiltration of adipose tissue persisted, notably these cells were less pro-inflammatory, had lower CXCL1 production, and consequently, lower neutrophil chemoattraction into the tissue. These findings reveal macrophage NOD1 as a cell-specific target to combat diet-induced inflammation past the step of macrophage infiltration, leading to insulin resistance.

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Teaser

Chan et al. identify that activators of NOD1, a receptor for bacterial cell wall peptidoglycan, increase in the bloodstream during high fat feeding. Moreover, depleting NOD1 from the immune system prevents pro-inflammatory macrophage activation and neutrophil infiltration in adipose tissue during a high fat diet, to improve whole-body insulin sensitivity.


http://ift.tt/2lV3p29

CD95/Fas Increases Stemness in Cancer Cells by Inducing a STAT1-Dependent Type I Interferon Response

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Abdul S. Qadir, Paolo Ceppi, Sonia Brockway, Calvin Law, Liang Mu, Nikolai N. Khodarev, Jung Kim, Jonathan C. Zhao, William Putzbach, Andrea E. Murmann, Zhuo Chen, Wenjing Chen, Xia Liu, Arthur R. Salomon, Huiping Liu, Ralph R. Weichselbaum, Jindan Yu, Marcus E. Peter
Stimulation of CD95/Fas drives and maintains cancer stem cells (CSCs). We now report that this involves activation of signal transducer and activator of transcription 1 (STAT1) and induction of STAT1-regulated genes and that this process is inhibited by active caspases. STAT1 is enriched in CSCs in cancer cell lines, patient-derived human breast cancer, and CD95^high-expressing glioblastoma neurospheres. CD95 stimulation of cancer cells induced secretion of type I interferons (IFNs) that bind to type I IFN receptors, resulting in activation of Janus-activated kinases, activation of STAT1, and induction of a number of STAT1-regulated genes that are part of a gene signature recently linked to therapy resistance in five primary human cancers. Consequently, we identified type I IFNs as drivers of cancer stemness. Knockdown or knockout of STAT1 resulted in a strongly reduced ability of CD95L or type I IFN to increase cancer stemness. This identifies STAT1 as a key regulator of the CSC-inducing activity of CD95.

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Teaser

Chronic stimulation of the death receptor CD95/Fas by CD95 ligand induces cancer stemness. Qadir et al. report that this activity involves induction of type I interferons followed by activation of STAT1 and Janus kinases downstream of the type I interferon receptors.


http://ift.tt/2lVbKCX

MAP3K4 Controls the Chromatin Modifier HDAC6 during Trophoblast Stem Cell Epithelial-to-Mesenchymal Transition

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Robert J. Mobley, Deepthi Raghu, Lauren D. Duke, Kayley Abell-Hart, Jon S. Zawistowski, Kyla Lutz, Shawn M. Gomez, Sujoy Roy, Ramin Homayouni, Gary L. Johnson, Amy N. Abell
The first epithelial-to-mesenchymal transition (EMT) occurs in trophoblast stem (TS) cells during implantation. Inactivation of the serine/threonine kinase MAP3K4 in TS cells (TS^KI4 cells) induces an intermediate state of EMT, where cells retain stemness, lose epithelial markers, and gain mesenchymal characteristics. Investigation of relationships among MAP3K4 activity, stemness, and EMT in TS cells may reveal key regulators of EMT. Here, we show that MAP3K4 activity controls EMT through the ubiquitination and degradation of HDAC6. Loss of MAP3K4 activity in TS^KI4 cells results in elevated HDAC6 expression and the deacetylation of cytoplasmic and nuclear targets. In the nucleus, HDAC6 deacetylates the promoters of tight junction genes, promoting the dissolution of tight junctions. Importantly, HDAC6 knockdown in TS^KI4 cells restores epithelial features, including cell-cell adhesion and barrier formation. These data define a role for HDAC6 in regulating gene expression during transitions between epithelial and mesenchymal phenotypes.

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Teaser

Mobley et al. find that HDAC6 is regulated by MAP3K4 during trophoblast stem cell differentiation and EMT. MAP3K4 promotes HDAC6 ubiquitination and degradation, maintaining the epithelial state. During EMT, HDAC6 directly deacetylates histones on epithelial gene promoters such as claudin6 and occludin, promoting the dissolution of tight junctions.


http://ift.tt/2lVewrQ

A Bach2-Cebp Gene Regulatory Network for the Commitment of Multipotent Hematopoietic Progenitors

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Ari Itoh-Nakadai, Mitsuyo Matsumoto, Hiroki Kato, Junichi Sasaki, Yukihiro Uehara, Yuki Sato, Risa Ebina-Shibuya, Mizuho Morooka, Ryo Funayama, Keiko Nakayama, Kyoko Ochiai, Akihiko Muto, Kazuhiko Igarashi
Hematopoietic stem cell and multipotent progenitor (MPP) commitment can be tuned in response to an infection so that their differentiation is biased toward myeloid cells. Here, we find that Bach2, which inhibits myeloid differentiation in common lymphoid progenitors, represses a cohort of myeloid genes and activates those linked to lymphoid function. Bach2 repressed both Cebpb and its target Csf1r, encoding C/EBPβ and macrophage colony-stimulating factor receptor (M-CSFr), respectively, whereas C/EBPβ repressed Bach2 and activated Csf1r. Bach2 and C/EBPβ further bound to overlapping regulatory regions at their myeloid target genes, suggesting the presence of a gene regulatory network (GRN) with mutual repression between these factors and a feedforward loop leading to myeloid gene regulation. Lipopolysaccharide reduced the expression of Bach2, resulting in enhanced myeloid differentiation. The Bach2-C/EBPβ GRN pathway thus tunes MPP commitment to myeloid and lymphoid lineages both under normal conditions and after infection.

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Microbes skew the balance of innate and acquired immune cells. Itoh-Nakadai et al. report that Bach2 and C/EBP form a gene regulatory network with mutual repression and antagonistic, feedforward regulation of myeloid genes. Bach2 tunes the commitment of multipotent progenitors to myeloid and lymphoid lineages under both normal and infectious conditions.


http://ift.tt/2lVpuxp

Macrophage Death following Influenza Vaccination Initiates the Inflammatory Response that Promotes Dendritic Cell Function in the Draining Lymph Node

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Nikolaos Chatziandreou, Yagmur Farsakoglu, Miguel Palomino-Segura, Rocco D'Antuono, Diego Ulisse Pizzagalli, Federica Sallusto, Veronika Lukacs-Kornek, Mariagrazia Uguccioni, Davide Corti, Shannon J. Turley, Antonio Lanzavecchia, Michael C. Carroll, Santiago F. Gonzalez
The mechanism by which inflammation influences the adaptive response to vaccines is not fully understood. Here, we examine the role of lymph node macrophages (LNMs) in the induction of the cytokine storm triggered by inactivated influenza virus vaccine. Following vaccination, LNMs undergo inflammasome-independent necrosis-like death that is reliant on MyD88 and Toll-like receptor 7 (TLR7) expression and releases pre-stored interleukin-1α (IL-1α). Furthermore, activated medullary macrophages produce interferon-β (IFN-β) that induces the autocrine secretion of IL-1α. We also found that macrophage depletion promotes lymph node-resident dendritic cell (LNDC) relocation and affects the capacity of CD11b⁺ LNDCs to capture virus and express co-stimulatory molecules. Inhibition of the IL-1α-induced inflammatory cascade reduced B cell responses, while co-administration of recombinant IL-1α increased the humoral response. Stimulation of the IL-1α inflammatory pathway might therefore represent a strategy to enhance antigen presentation by LNDCs and improve the humoral response against influenza vaccines.

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Teaser

The mechanism by which inflammation influences the antibody response to vaccines is unclear. Chatziandreou et al. found that lymph node macrophages are key players in the initiation of the IL-1α-mediated inflammatory response that follows influenza vaccination.


http://ift.tt/2lVewbk

Distinct Kinase-Independent Role of RIPK3 in CD11c+ Mononuclear Phagocytes in Cytokine-Induced Tissue Repair

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Kenta Moriwaki, Sakthi Balaji, John Bertin, Peter J. Gough, Francis Ka-Ming Chan
Receptor interacting protein kinase 3 (RIPK3) induces necroptosis, a type of regulated necrosis, through its kinase domain and receptor interacting protein (RIP) homotypic interaction motif (RHIM). In addition, RIPK3 has been shown to regulate NLRP3 inflammasome and nuclear factor κB (NF-κB) activation. However, the relative contribution of these signaling pathways to RIPK3-dependent inflammation in distinct immune effectors is unknown. To investigate these questions, we generated RIPK3-GFP reporter mice. We found that colonic CD11c⁺CD11b⁺CD14⁺ mononuclear phagocytes (MNPs) expressed the highest level of RIPK3 in the lamina propria. Consequently, deletion of the RIPK3 RHIM in CD11c⁺ cells alone was sufficient to impair dextran sodium sulfate (DSS)-induced interleukin (IL)-23 and IL-1β expression, leading to severe intestinal inflammation. In contrast, mice expressing kinase inactive RIPK3 were not hypersensitive to DSS. Thus, a key physiological function of RIPK3 is to promote reparative cytokine expression through intestinal CD11c⁺ MNPs in a kinase- and necroptosis-independent manner.

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Teaser

Moriwaki et al. demonstrate that RIPK3 promotes cytokine production in CD11c⁺ mononuclear phagocytes in an RHIM-dependent, but kinase-independent manner. This necroptosis-independent function of RIPK3 is crucial for tissue repair in response to intestinal injury.


http://ift.tt/2lVkuZT

Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Swagata Dey, Gary Banker, Krishanu Ray
Local endosomal recycling at synapses is essential to maintain neurotransmission. Rab4GTPase, found on sorting endosomes, is proposed to balance the flow of vesicles among endocytic, recycling, and degradative pathways in the presynaptic compartment. Here, we report that Rab4-associated vesicles move bidirectionally in Drosophila axons but with an anterograde bias, resulting in their moderate enrichment at the synaptic region of the larval ventral ganglion. Results from FK506 binding protein (FKBP) and FKBP-Rapamycin binding domain (FRB) conjugation assays in rat embryonic fibroblasts together with genetic analyses in Drosophila indicate that an association with Kinesin-2 (mediated by the tail domain of Kinesin-2α/KIF3A/KLP64D subunit) moves Rab4-associated vesicles toward the synapse. Reduction in the anterograde traffic of Rab4 causes an expansion of the volume of the synapse-bearing region in the ventral ganglion and increases the motility of Drosophila larvae. These results suggest that Rab4-dependent vesicular traffic toward the synapse plays a vital role in maintaining synaptic balance in this neuronal network.

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Teaser

Dey et al. show that in Drosophila larvae, binding to the Kinesin-2α tail propels Rab4-associated vesicles toward the synapse. Reduced Rab4 transport expands the synapse-bearing region of the ventral ganglion and enhances larval motility. Hence, Kinesin-2-mediated Rab4 trafficking appears to regulate synapse homeostasis in a neuronal network.


http://ift.tt/2lVe2SA

Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Sayan Chakraborty, Kizito Njah, Ajaybabu V. Pobbati, Ying Bena Lim, Anandhkumar Raju, Manikandan Lakshmanan, Vinay Tergaonkar, Chwee Teck Lim, Wanjin Hong
The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. Here, we uncover a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 signaling and negating the functions of Merlin and LATS1/2. We further show that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. We propose that Agrin acts as a mechanotransduction signal in the ECM.

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Teaser

Chakraborty et al. report that the extracellular matrix protein Agrin is a mechanotransducing signal activating YAP through the integrin-focal adhesion-Lrp4/MuSK receptor pathway. Agrin signals matrix and cellular rigidity by activating FAK-ILK-PAK1 signaling that negates the Hippo tumor-suppressor pathway. Importantly, Agrin relies on YAP for oncogenic activities underlying liver cancer.


http://ift.tt/2lVidxQ

Integrin Beta 3 Regulates Cellular Senescence by Activating the TGF-β Pathway

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Valentina Rapisarda, Michela Borghesan, Veronica Miguela, Vesela Encheva, Ambrosius P. Snijders, Amaia Lujambio, Ana O'Loghlen
Cellular senescence is an important in vivo mechanism that prevents the propagation of damaged cells. However, the precise mechanisms regulating senescence are not well characterized. Here, we find that ITGB3 (integrin beta 3 or β3) is regulated by the Polycomb protein CBX7. β3 expression accelerates the onset of senescence in human primary fibroblasts by activating the transforming growth factor β (TGF-β) pathway in a cell-autonomous and non-cell-autonomous manner. β3 levels are dynamically increased during oncogene-induced senescence (OIS) through CBX7 Polycomb regulation, and downregulation of β3 levels overrides OIS and therapy-induced senescence (TIS), independently of its ligand-binding activity. Moreover, cilengitide, an αvβ3 antagonist, has the ability to block the senescence-associated secretory phenotype (SASP) without affecting proliferation. Finally, we show an increase in β3 levels in a subset of tissues during aging. Altogether, our data show that integrin β3 subunit is a marker and regulator of senescence.

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Teaser

Rapisarda et al. show that integrin β3 subunit expression induces senescence by activating TGF-β, while β3 knockdown overcomes senescence. β3 is dynamically upregulated in OIS and has ligand-independent activity. They also find a positive correlation between β3 levels and aging in a subset of tissues.


http://ift.tt/2lVpvkV

Negative Regulators of an RNAi-Heterochromatin Positive Feedback Loop Safeguard Somatic Genome Integrity in Tetrahymena

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Jan H. Suhren, Tomoko Noto, Kensuke Kataoka, Shan Gao, Yifan Liu, Kazufumi Mochizuki
RNAi-mediated positive feedback loops are pivotal for the maintenance of heterochromatin, but how they are downregulated at heterochromatin-euchromatin borders is not well understood. In the ciliated protozoan Tetrahymena, heterochromatin is formed exclusively on the sequences that are removed from the somatic genome by programmed DNA elimination, and an RNAi-mediated feedback loop is important for assembling heterochromatin on the eliminated sequences. In this study, we show that the heterochromatin protein 1 (HP1)-like protein Coi6p, its interaction partners Coi7p and Lia5p, and the histone demethylase Jmj1p are crucial for confining the production of small RNAs and the formation of heterochromatin to the eliminated sequences. The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. The results provide direct evidence for the existence of a dedicated mechanism that counteracts a positive feedback loop between RNAi and heterochromatin at heterochromatin-euchromatin borders to maintain the integrity of the somatic genome.

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Teaser

Regulation of a small RNA-mediated positive feedback loop is pivotal for confining heterochromatin to proper locations. Suhren et al. show the existence of a dedicated mechanism that counteracts a small RNA-heterochromatin positive feedback loop at heterochromatin-euchromatin borders to maintain the integrity of the somatic genome in Tetrahymena.


http://ift.tt/2lVgoRe

Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Robert C. Alver, Gaganmeet Singh Chadha, Peter J. Gillespie, J. Julian Blow
Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation.

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Teaser

Alver et al. find that DDK-dependent MCM2-7 phosphorylation is opposed by Rif1-targeted PP1. Lack of Rif1 increases MCM phosphorylation and abolishes the ability to block origin firing, even when undergoing replication stress. In the absence of CHK1, the stability of stalled replisomes is severely compromised upon Rif-PP1-mediated dephosphorylation.


http://ift.tt/2lVcLek

Unsupervised Spike Sorting for Large-Scale, High-Density Multielectrode Arrays

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Gerrit Hilgen, Martino Sorbaro, Sahar Pirmoradian, Jens-Oliver Muthmann, Ibolya Edit Kepiro, Simona Ullo, Cesar Juarez Ramirez, Albert Puente Encinas, Alessandro Maccione, Luca Berdondini, Vittorio Murino, Diego Sona, Francesca Cella Zanacchi, Evelyne Sernagor, Matthias Helge Hennig
We present a method for automated spike sorting for recordings with high-density, large-scale multielectrode arrays. Exploiting the dense sampling of single neurons by multiple electrodes, an efficient, low-dimensional representation of detected spikes consisting of estimated spatial spike locations and dominant spike shape features is exploited for fast and reliable clustering into single units. Millions of events can be sorted in minutes, and the method is parallelized and scales better than quadratically with the number of detected spikes. Performance is demonstrated using recordings with a 4,096-channel array and validated using anatomical imaging, optogenetic stimulation, and model-based quality control. A comparison with semi-automated, shape-based spike sorting exposes significant limitations of conventional methods. Our approach demonstrates that it is feasible to reliably isolate the activity of up to thousands of neurons and that dense, multi-channel probes substantially aid reliable spike sorting.

Graphical abstract

Teaser

Data volume and complexity make spike sorting for large-scale extracellular recordings computationally extremely challenging. Hilgen et al. introduce a method enabling analysis of recordings with thousands of channels and provide tools for automated quality control and unit selection.


http://ift.tt/2lVfR1O

A Knockin Reporter Allows Purification and Characterization of mDA Neurons from Heterogeneous Populations

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Ninuo Xia, Fang Fang, Pengbo Zhang, Jun Cui, Chhavy Tep-Cullison, Tim Hamerley, Hyun Joo Lee, Theo Palmer, Brian Bothner, Jin Hyung Lee, Renee Reijo Pera
Generation of midbrain dopaminergic (mDA) neurons from human pluripotent stem cells provides a platform for inquiry into basic and translational studies of Parkinson's disease (PD). However, heterogeneity in differentiation in vitro makes it difficult to identify mDA neurons in culture or in vivo following transplantation. Here, we report the generation of a human embryonic stem cell (hESC) line with a tyrosine hydroxylase (TH)-RFP (red fluorescent protein) reporter. We validated that RFP faithfully mimicked TH expression during differentiation. Use of this TH-RFP reporter cell line enabled purification of mDA-like neurons from heterogeneous cultures with subsequent characterization of neuron transcriptional and epigenetic programs (global binding profiles of H3K27ac, H3K4me1, and 5-hydroxymethylcytosine [5hmC]) at four different stages of development. We anticipate that the tools and data described here will contribute to the development of mDA neurons for applications in disease modeling and/or drug screening and cell replacement therapies for PD.

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Teaser

Xia et al. create a knockin human tyrosine hydroxylase (TH) reporter line to isolate TH-positive dopaminergic neurons from heterogeneous population and examine their transcriptional landscape.


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