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Full-Text Articles in Cell Biology

Endothelial-Specific Inhibition Of Nf-Kappab Enhances Functional Haematopoiesis, Michael G. Poulos, Pradeep Ramalingam, Michael C. Gutkin, Maria Kleppe, Michael Ginsberg, Michael J. P. Crowley, Olivier Elemento, Ross L. Levine, Shahin Rafii, Jan Kitajewski, Matthew B. Greenblatt, Jae-Hyuck Shim, Jason M. Butler Dec 2016

Endothelial-Specific Inhibition Of Nf-Kappab Enhances Functional Haematopoiesis, Michael G. Poulos, Pradeep Ramalingam, Michael C. Gutkin, Maria Kleppe, Michael Ginsberg, Michael J. P. Crowley, Olivier Elemento, Ross L. Levine, Shahin Rafii, Jan Kitajewski, Matthew B. Greenblatt, Jae-Hyuck Shim, Jason M. Butler

Open Access Articles

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-kappaB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-kappaB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IkappaBalpha cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated ...


Consensus Paper: Cerebellar Development, Ketty Leto, Baojin Ding, Daniel Lee Kilpatrick Dec 2016

Consensus Paper: Cerebellar Development, Ketty Leto, Baojin Ding, Daniel Lee Kilpatrick

Open Access Articles

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.


The Mammalian Linc Complex Regulates Genome Transcriptional Responses To Substrate Rigidity, Samer G. Alam, Qiao Zhang, Nripesh Prasad, Yuan Li, Srikar Chamala, Ram Kuchibhotla, Birendra Kc, Varun Aggarwal, Shristi Shrestha, Angela L. Jones, Shawn E. Levy, Kyle J. Roux, Jeffrey A. Nickerson, Tanmay P. Lele Dec 2016

The Mammalian Linc Complex Regulates Genome Transcriptional Responses To Substrate Rigidity, Samer G. Alam, Qiao Zhang, Nripesh Prasad, Yuan Li, Srikar Chamala, Ram Kuchibhotla, Birendra Kc, Varun Aggarwal, Shristi Shrestha, Angela L. Jones, Shawn E. Levy, Kyle J. Roux, Jeffrey A. Nickerson, Tanmay P. Lele

Open Access Articles

Mechanical integration of the nucleus with the extracellular matrix (ECM) is established by linkage between the cytoskeleton and the nucleus. This integration is hypothesized to mediate sensing of ECM rigidity, but parsing the function of nucleus-cytoskeleton linkage from other mechanisms has remained a central challenge. Here we took advantage of the fact that the LINC (linker of nucleoskeleton and cytoskeleton) complex is a known molecular linker of the nucleus to the cytoskeleton, and asked how it regulates the sensitivity of genome-wide transcription to substratum rigidity. We show that gene mechanosensitivity is preserved after LINC disruption, but reversed in direction. Combined ...


Adipose-Derived Human Stem/Stromal Cells: Comparative Organ Specific Mitochondrial Bioenergy Profiles, Alice S. Ferng, Katherine M. Marsh, Jamie M. Fleming, Renee F. Conway, David Schipper, Naing Bajaj, Alana M. Connell, Tia Pilikian, Kitsie Johnson, Ray Runyan, Stephen M. Black, John A. Szivek, Zain Khalpey Dec 2016

Adipose-Derived Human Stem/Stromal Cells: Comparative Organ Specific Mitochondrial Bioenergy Profiles, Alice S. Ferng, Katherine M. Marsh, Jamie M. Fleming, Renee F. Conway, David Schipper, Naing Bajaj, Alana M. Connell, Tia Pilikian, Kitsie Johnson, Ray Runyan, Stephen M. Black, John A. Szivek, Zain Khalpey

Open Access Articles

BACKGROUND: Adipose-derived stem/stromal cells (ASCs) isolated from the stromal vascular fraction are a source of mesenchymal stem cells that have been shown to be beneficial in many regenerative medicine applications. ASCs are an attractive source of stem cells in particular, due to their lack of immunogenicity. This study examines differences between mitochondrial bioenergetic profiles of ASCs isolated from adipose tissue of five peri-organ regions: pericardial, thymic, knee, shoulder, and abdomen.

RESULTS: Flow cytometry showed that the majority of each ASC population isolated from the adipose tissue of 12 donors, with an n = 3 for each tissue type, were positive ...


High-Resolution Proteomic And Lipidomic Analysis Of Exosomes And Microvesicles From Different Cell Sources, Reka A. Haraszti, Marie-Cecile Didiot, Ellen Sapp, John D. Leszyk, Scott A. Shaffer, Hannah E. Rockwell, Fei Gao, Niven R. Narain, Marian Difiglia, Michael A. Kiebish, Neil Aronin, Anastasia Khvorova Nov 2016

High-Resolution Proteomic And Lipidomic Analysis Of Exosomes And Microvesicles From Different Cell Sources, Reka A. Haraszti, Marie-Cecile Didiot, Ellen Sapp, John D. Leszyk, Scott A. Shaffer, Hannah E. Rockwell, Fei Gao, Niven R. Narain, Marian Difiglia, Michael A. Kiebish, Neil Aronin, Anastasia Khvorova

Open Access Articles

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were ...


Dna Methylation Directs Genomic Localization Of Mbd2 And Mbd3 In Embryonic Stem Cells, Sarah J. Hainer, Kurtis N. Mccannell, Jun Yu, Ly-Sha Ee, Lihua (Julie) Zhu, Oliver J. Rando, Thomas G. Fazzio Nov 2016

Dna Methylation Directs Genomic Localization Of Mbd2 And Mbd3 In Embryonic Stem Cells, Sarah J. Hainer, Kurtis N. Mccannell, Jun Yu, Ly-Sha Ee, Lihua (Julie) Zhu, Oliver J. Rando, Thomas G. Fazzio

Open Access Articles

Cytosine methylation is an epigenetic and regulatory mark that functions in part through recruitment of chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins. Two MBD proteins, Mbd2 and Mbd3, were previously shown to bind methylated or hydroxymethylated DNA, respectively; however, both of these findings have been disputed. Here, we investigated this controversy using experimental approaches and re-analysis of published data and find no evidence for methylation-independent functions of Mbd2 or Mbd3. We show that chromatin localization of Mbd2 and Mbd3 is highly overlapping and, unexpectedly, we find Mbd2 and Mbd3 are interdependent for chromatin association. Further investigation reveals that ...


One-Step Immortalization Of Primary Human Airway Epithelial Cells Capable Of Oncogenic Transformation, Jordan L. Smith, Liam C. Lee, Abigail Read, Qiuning Li, Bing Yu, Chih-Shia Lee, Ji Luo Nov 2016

One-Step Immortalization Of Primary Human Airway Epithelial Cells Capable Of Oncogenic Transformation, Jordan L. Smith, Liam C. Lee, Abigail Read, Qiuning Li, Bing Yu, Chih-Shia Lee, Ji Luo

Open Access Articles

BACKGROUND: The ability to transform normal human cells into cancer cells with the introduction of defined genetic alterations is a valuable method for understanding the mechanisms of oncogenesis. Easy establishment of immortalized but non-transformed human cells from various tissues would facilitate these genetic analyses.

RESULTS: We report here a simple, one-step immortalization method that involves retroviral vector mediated co-expression of the human telomerase protein and a shRNA targeting the CDKN2A gene locus. We demonstrate that this method could successfully immortalize human small airway epithelial cells while maintaining their chromosomal stability. We further showed that these cells retain p53 activity and ...


Transient Runx1 Expression During Early Mesendodermal Differentiation Of Hescs Promotes Epithelial To Mesenchymal Transition Through Tgfb2 Signaling, Jennifer J. Vanoudenhove, Ricardo F. Medina, Prachi N. Ghule University Of Vermont College Of Medicine, Jane B. Lian, Janet L. Stein, Sayyed K. Zaidi, Gary S. Stein Nov 2016

Transient Runx1 Expression During Early Mesendodermal Differentiation Of Hescs Promotes Epithelial To Mesenchymal Transition Through Tgfb2 Signaling, Jennifer J. Vanoudenhove, Ricardo F. Medina, Prachi N. Ghule University Of Vermont College Of Medicine, Jane B. Lian, Janet L. Stein, Sayyed K. Zaidi, Gary S. Stein

Open Access Articles

The transition of human embryonic stem cells (hESCs) from pluripotency to lineage commitment is not fully understood, and a role for phenotypic transcription factors in the initial stages of hESC differentiation remains to be explored. From a screen of candidate factors, we found that RUNX1 is selectively and transiently upregulated early in hESC differentiation to mesendodermal lineages. Transcriptome profiling and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that loss of RUNX1 impaired an epithelial to mesenchymal transition during differentiation. Cell ...


A Novel Protocol For Directed Differentiation Of C9orf72-Associated Human Induced Pluripotent Stem Cells Into Contractile Skeletal Myotubes, Elliot W. Swartz, Jaeyun Baek, Mochtar Pribadi, Kevin J. Wojta, Sandra Almeida, Anna M. Karydas, Fen-Biao Gao, Bruce L. Miller, Giovanni Coppola Nov 2016

A Novel Protocol For Directed Differentiation Of C9orf72-Associated Human Induced Pluripotent Stem Cells Into Contractile Skeletal Myotubes, Elliot W. Swartz, Jaeyun Baek, Mochtar Pribadi, Kevin J. Wojta, Sandra Almeida, Anna M. Karydas, Fen-Biao Gao, Bruce L. Miller, Giovanni Coppola

Open Access Articles

Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to be used for the study of underlying molecular biology of disease, therapeutic drug screening, and transplant-based regenerative medicine. However, methods for the directed differentiation of skeletal muscle for these purposes remain scarce and incomplete. Here, we present a novel, small molecule-based protocol for the generation of multinucleated skeletal myotubes using eight independent iPSC lines. Through combinatorial inhibition of phosphoinositide 3-kinase (PI3K) and glycogen synthase kinase 3beta (GSK3beta) with addition of bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2), we report up to 64% conversion of ...


The Rna-Binding Protein Atx-2 Regulates Cytokinesis Through Par-5 And Zen-4, Megan M. Gnazzo, Eva-Maria E. Uhlemann, Alex R. Villarreal, Masaki Shirayama, Eddie G. Dominguez, Ahna R. Skop Oct 2016

The Rna-Binding Protein Atx-2 Regulates Cytokinesis Through Par-5 And Zen-4, Megan M. Gnazzo, Eva-Maria E. Uhlemann, Alex R. Villarreal, Masaki Shirayama, Eddie G. Dominguez, Ahna R. Skop

Open Access Articles

The spindle midzone harbors both microtubules and proteins necessary for furrow formation and the completion of cytokinesis. However, the mechanisms that mediate the temporal and spatial recruitment of cell division factors to the spindle midzone and midbody remain unclear. Here we describe a mechanism governed by the conserved RNA-binding protein ATX-2/Ataxin-2, which targets and maintains ZEN-4 at the spindle midzone. ATX-2 does this by regulating the amount of PAR-5 at mitotic structures, particularly the spindle, centrosomes, and midbody. Preventing ATX-2 function leads to elevated levels of PAR-5, enhanced chromatin and centrosome localization of PAR-5-GFP, and ultimately a reduction of ...


A Widely Employed Germ Cell Marker Is An Ancient Disordered Protein With Reproductive Functions In Diverse Eukaryotes, Michelle A. Carmell, Gregoriy A. Dokshin, Helen Skaletsky, Yueh-Chiang Hu, Josien C. Van Wolfswinkel, Kyomi J. Igarashi, Daniel W. Bellott, Michael Nefedov, Peter W. Reddien, George C. Enders, Vladimir N. Uversky, Craig C. Mello, David C. Page Oct 2016

A Widely Employed Germ Cell Marker Is An Ancient Disordered Protein With Reproductive Functions In Diverse Eukaryotes, Michelle A. Carmell, Gregoriy A. Dokshin, Helen Skaletsky, Yueh-Chiang Hu, Josien C. Van Wolfswinkel, Kyomi J. Igarashi, Daniel W. Bellott, Michael Nefedov, Peter W. Reddien, George C. Enders, Vladimir N. Uversky, Craig C. Mello, David C. Page

Open Access Articles

The advent of sexual reproduction and the evolution of a dedicated germline in multicellular organisms are critical landmarks in eukaryotic evolution. We report an ancient family of GCNA (germ cell nuclear antigen) proteins that arose in the earliest eukaryotes, and feature a rapidly evolving intrinsically disordered region (IDR). Phylogenetic analysis reveals that GCNA proteins emerged before the major eukaryotic lineages diverged; GCNA predates the origin of a dedicated germline by a billion years. Gcna gene expression is enriched in reproductive cells across eukarya - either just prior to or during meiosis in single-celled eukaryotes, and in stem cells and germ cells ...


The Zn-Finger Domain Of Mdmx Suppresses Cancer Progression By Promoting Genome Stability In P53-Mutant Cells, Zdenka Matijasevic, Anna Krzywicka-Racka, Greenfield Sluder, Judith Gallant, Stephen N. Jones Oct 2016

The Zn-Finger Domain Of Mdmx Suppresses Cancer Progression By Promoting Genome Stability In P53-Mutant Cells, Zdenka Matijasevic, Anna Krzywicka-Racka, Greenfield Sluder, Judith Gallant, Stephen N. Jones

Open Access Articles

The MDMX (MDM4) oncogene is amplified or overexpressed in a significant percentage of human tumors. MDMX is thought to function as an oncoprotein by binding p53 tumor suppressor protein to inhibit p53-mediated transcription, and by complexing with MDM2 oncoprotein to promote MDM2-mediated degradation of p53. However, down-regulation or loss of functional MDMX has also been observed in a variety of human tumors that are mutated for p53, often correlating with more aggressive cancers and a worse patient prognosis. We have previously reported that endogenous levels of MdmX can suppress proliferation and promote pseudo-bipolar mitosis in primary and tumor cells derived ...


Autophagy Activation By Transcription Factor Eb (Tfeb) In Striatum Of Hdq175/Q7 Mice, Petr Vodicka, Kathryn O. Chase, Maria Iuliano, Adelaide Tousley, Dana T. Valentine, Ellen Sapp, Kimberly B. Kegel-Gleason, Miguel Sena-Esteves, Neil Aronin, Marian Difiglia Oct 2016

Autophagy Activation By Transcription Factor Eb (Tfeb) In Striatum Of Hdq175/Q7 Mice, Petr Vodicka, Kathryn O. Chase, Maria Iuliano, Adelaide Tousley, Dana T. Valentine, Ellen Sapp, Kimberly B. Kegel-Gleason, Miguel Sena-Esteves, Neil Aronin, Marian Difiglia

Open Access Articles

BACKGROUND: Mutant huntingtin (mHTT) is encoded by the Huntington's disease (HD) gene and its accumulation in the brain contributes to HD pathogenesis. Reducing mHTT levels through activation of the autophagosome-lysosomal pathway may have therapeutic benefit. Transcription factor EB (TFEB) regulates lysosome biogenesis and autophagy.

OBJECTIVE: To examine if increasing TFEB protein levels in HD mouse striatum induces autophagy and influences mHTT levels.

METHODS: We introduced cDNA encoding TFEB with an HA tag (TFEB-HA) under the control of neuron specific synapsin 1 promoter into the striatum of 3 month old HDQ175/Q7 mice using adeno-associated virus AAV2/9. The levels ...


Adipocyte-Specific Hypoxia-Inducible Gene 2 Promotes Fat Deposition And Diet-Induced Insulin Resistance, Marina Distefano, Rachel J. Roth Flach, Ozlem Senol-Cosar, Laura V. Danai, Joseph V. Virbasius, Sarah M. Nicoloro, Juerg R. Straubhaar, Sezin Dagdeviren, Martin Wabitsch, Olga T. Gupta, Jason K. Kim, Michael P. Czech Sep 2016

Adipocyte-Specific Hypoxia-Inducible Gene 2 Promotes Fat Deposition And Diet-Induced Insulin Resistance, Marina Distefano, Rachel J. Roth Flach, Ozlem Senol-Cosar, Laura V. Danai, Joseph V. Virbasius, Sarah M. Nicoloro, Juerg R. Straubhaar, Sezin Dagdeviren, Martin Wabitsch, Olga T. Gupta, Jason K. Kim, Michael P. Czech

Open Access Articles

OBJECTIVE: Adipose tissue relies on lipid droplet (LD) proteins in its role as a lipid-storing endocrine organ that controls whole body metabolism. Hypoxia-inducible Gene 2 (Hig2) is a recently identified LD-associated protein in hepatocytes that promotes hepatic lipid storage, but its role in the adipocyte had not been investigated. Here we tested the hypothesis that Hig2 localization to LDs in adipocytes promotes adipose tissue lipid deposition and systemic glucose homeostasis.

METHOD: White and brown adipocyte-deficient (Hig2fl/fl x Adiponection cre+) and selective brown/beige adipocyte-deficient (Hig2fl/fl x Ucp1 cre+) mice were generated to investigate the role of Hig2 in ...


Regulation Of X-Linked Gene Expression During Early Mouse Development By Rlim, Feng Wang, Jongdae Shin, Jeremy Shea, Jun Yu, Ana Boskovic, Meg Byron, Xiaochun Zhu, Alex K. Shalek, Aviv Regev, Jeanne B. Lawrence, Eduardo M. Torres, Lihua J. Zhu, Oliver J. Rando, Ingolf Bach Sep 2016

Regulation Of X-Linked Gene Expression During Early Mouse Development By Rlim, Feng Wang, Jongdae Shin, Jeremy Shea, Jun Yu, Ana Boskovic, Meg Byron, Xiaochun Zhu, Alex K. Shalek, Aviv Regev, Jeanne B. Lawrence, Eduardo M. Torres, Lihua J. Zhu, Oliver J. Rando, Ingolf Bach

Open Access Articles

Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our ...


Mmp-9 And Mmp-2 Contribute To Neuronal Cell Death In Ipsc Models Of Frontotemporal Dementia With Mapt Mutations, Md Helal Uddin Biswas, Sandra Almeida, Rodrigo Lopez-Gonzalez, Wenjie Mao, Zhijun Zhang, Anna M. Karydas, Michael D. Geschwind, Jacek Biernat, Eva-Maria Mandelkow, Kensuke Futai, Bruce L. Miller, Fen-Biao Gao Sep 2016

Mmp-9 And Mmp-2 Contribute To Neuronal Cell Death In Ipsc Models Of Frontotemporal Dementia With Mapt Mutations, Md Helal Uddin Biswas, Sandra Almeida, Rodrigo Lopez-Gonzalez, Wenjie Mao, Zhijun Zhang, Anna M. Karydas, Michael D. Geschwind, Jacek Biernat, Eva-Maria Mandelkow, Kensuke Futai, Bruce L. Miller, Fen-Biao Gao

Open Access Articles

How mutations in the microtubule-associated protein tau (MAPT) gene cause frontotemporal dementia (FTD) remains poorly understood. We generated and characterized multiple induced pluripotent stem cell (iPSC) lines from patients with MAPT IVS10+16 and tau-A152T mutations and a control subject. In cortical neurons differentiated from these and other published iPSC lines, we found that MAPT mutations do not affect neuronal differentiation but increase the 4R/3R tau ratio. Patient neurons had significantly higher levels of MMP-9 and MMP-2 and were more sensitive to stress-induced cell death. Inhibitors of MMP-9/MMP-2 protected patient neurons from stress-induced cell death and recombinant MMP-9 ...


Human Ipsc-Derived Neuronal Model Of Tau-A152t Frontotemporal Dementia Reveals Tau-Mediated Mechanisms Of Neuronal Vulnerability, M. Catarina Silva, Sandra Almeida, Md Helal Uddin Biswas, Zhijun Zhang, Fen-Biao Gao, Stephen J. Haggarty Sep 2016

Human Ipsc-Derived Neuronal Model Of Tau-A152t Frontotemporal Dementia Reveals Tau-Mediated Mechanisms Of Neuronal Vulnerability, M. Catarina Silva, Sandra Almeida, Md Helal Uddin Biswas, Zhijun Zhang, Fen-Biao Gao, Stephen J. Haggarty

Open Access Articles

Frontotemporal dementia (FTD) and other tauopathies characterized by focal brain neurodegeneration and pathological accumulation of proteins are commonly associated with tau mutations. However, the mechanism of neuronal loss is not fully understood. To identify molecular events associated with tauopathy, we studied induced pluripotent stem cell (iPSC)-derived neurons from individuals carrying the tau-A152T variant. We highlight the potential of in-depth phenotyping of human neuronal cell models for pre-clinical studies and identification of modulators of endogenous tau toxicity. Through a panel of biochemical and cellular assays, A152T neurons showed accumulation, redistribution, and decreased solubility of tau. Upregulation of tau was coupled ...


Improved B Cell Development In Humanized Nod-Scid Il2rgammanull Mice Transgenically Expressing Human Stem Cell Factor, Granulocyte-Macrophage Colony-Stimulating Factor And Interleukin-3, Sonal Jangalwe, Leonard D. Shultz, Anuja Mathew, Michael A. Brehm Aug 2016

Improved B Cell Development In Humanized Nod-Scid Il2rgammanull Mice Transgenically Expressing Human Stem Cell Factor, Granulocyte-Macrophage Colony-Stimulating Factor And Interleukin-3, Sonal Jangalwe, Leonard D. Shultz, Anuja Mathew, Michael A. Brehm

Open Access Articles

INTRODUCTION: Immunodeficient mice engrafted with human immune systems support studies of human hematopoiesis and the immune response to human-specific pathogens. A significant limitation of these humanized mouse models is, however, a severely restricted ability of human B cells to undergo class switching and produce antigen-specific IgG after infection or immunization.

METHODS: In this study, we have characterized the development and function of human B cells in NOD-scid IL2Rgammanull (NSG) mice transgenically expressing human stem cell factor (SCF), granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-3 (NSG-SGM3) following engraftment with human hematopoietic stem cells, autologous fetal liver, and thymic tissues (bone marrow ...


Septate Junction Proteins Play Essential Roles In Morphogenesis Throughout Embryonic Development In Drosophila, Sonia Hall, Robert E. Ward 4th Aug 2016

Septate Junction Proteins Play Essential Roles In Morphogenesis Throughout Embryonic Development In Drosophila, Sonia Hall, Robert E. Ward 4th

Open Access Articles

The septate junction (SJ) is the occluding junction found in the ectodermal epithelia of invertebrate organisms, and is essential to maintain chemically distinct compartments in epithelial organs, to provide the blood-brain barrier in the nervous system, and to provide an important line of defense against invading pathogens. More than 20 genes have been identified to function in the establishment or maintenance of SJs in Drosophila melanogaster Numerous studies have demonstrated the cell biological function of these proteins in establishing the occluding junction, whereas very few studies have examined further developmental roles for them. Here we examined embryos with mutations in ...


The Histone H3k9 Demethylase Kdm3a Promotes Anoikis By Transcriptionally Activating Pro-Apoptotic Genes Bnip3 And Bnip3l, Victoria E. Pedanou, Stephane Gobeil, Sebastien Tabaries, Tessa M. Simone, Lihua Julie Zhu, Peter M. Siegel, Michael R. Green Jul 2016

The Histone H3k9 Demethylase Kdm3a Promotes Anoikis By Transcriptionally Activating Pro-Apoptotic Genes Bnip3 And Bnip3l, Victoria E. Pedanou, Stephane Gobeil, Sebastien Tabaries, Tessa M. Simone, Lihua Julie Zhu, Peter M. Siegel, Michael R. Green

Open Access Articles

Epithelial cells that lose attachment to the extracellular matrix undergo a specialized form of apoptosis called anoikis. Here, using large-scale RNA interference (RNAi) screening, we find that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase, plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. We find that KDM3A promotes anoikis through transcriptional activation ...


Levels Of Ycg1 Limit Condensin Function During The Cell Cycle, Tyler W. Doughty, Heather E. Arsenault, Jennifer A. Benanti Jul 2016

Levels Of Ycg1 Limit Condensin Function During The Cell Cycle, Tyler W. Doughty, Heather E. Arsenault, Jennifer A. Benanti

Open Access Articles

During mitosis chromosomes are condensed to facilitate their segregation, through a process mediated by the condensin complex. Although several factors that promote maximal condensin activity during mitosis have been identified, the mechanisms that downregulate condensin activity during interphase are largely unknown. Here, we demonstrate that Ycg1, the Cap-G subunit of budding yeast condensin, is cell cycle-regulated with levels peaking in mitosis and decreasing as cells enter G1 phase. This cyclical expression pattern is established by a combination of cell cycle-regulated transcription and constitutive degradation. Interestingly, overexpression of YCG1 and mutations that stabilize Ycg1 each result in delayed cell-cycle entry and ...


Epstein-Barr Virus Infection Of Mammary Epithelial Cells Promotes Malignant Transformation, Hai Hu, Joyce D. Fingeroth, Gerburg M. Wulf Jul 2016

Epstein-Barr Virus Infection Of Mammary Epithelial Cells Promotes Malignant Transformation, Hai Hu, Joyce D. Fingeroth, Gerburg M. Wulf

Open Access Articles

Whether the human tumor virus, Epstein-Barr Virus (EBV), promotes breast cancer remains controversial and a potential mechanism has remained elusive. Here we show that EBV can infect primary mammary epithelial cells (MECs) that express the receptor CD21. EBV infection leads to the expansion of early MEC progenitor cells with a stem cell phenotype, activates MET signaling and enforces a differentiation block. When MECs were implanted as xenografts, EBV infection cooperated with activated Ras and accelerated the formation of breast cancer. Infection in EBV-related tumors was of a latency type II pattern, similar to nasopharyngeal carcinoma (NPC). A human gene expression ...


Border Security: The Role Of Ripk3 In Epithelium Homeostasis, Kenta Moriwaki, Sakthi Balaji, Francis Ka-Ming Chan Jun 2016

Border Security: The Role Of Ripk3 In Epithelium Homeostasis, Kenta Moriwaki, Sakthi Balaji, Francis Ka-Ming Chan

Open Access Articles

Receptor interacting protein kinase 3 (RIPK3) is a crucial inducer of necroptosis. Its activity is controlled by interaction with other signal adaptors through the "RIP homotypic interaction motif" (RHIM). Recent studies revealed a critical function for RIPK3 in the maintenance of epithelial tissue integrity. In mice with genetic deficiency of the apoptosis adaptors FADD or caspase 8, RIPK3 promotes necroptotic cell death of epithelial cells, leading to excessive and lethal inflammation. In contrast, when FADD and caspase 8 functions are intact, RIPK3 serves as a protector of intestinal epithelial integrity by promoting injury-induced wound repair. In the latter case, RIPK3 ...


Cyld Proteolysis Protects Macrophages From Tnf-Mediated Auto-Necroptosis Induced By Lps And Licensed By Type I Ifn, Diana Legarda, Scott J. Justus, Rosalind L. Ang, Nimisha Rikhi, Wenjing Li, Thomas M. Moran, Jianke Zhang, Emiko Mizoguchi, Matija Zelic, Michelle A. Kelliher, J. Magarian Blander, Adrian T. Ting Jun 2016

Cyld Proteolysis Protects Macrophages From Tnf-Mediated Auto-Necroptosis Induced By Lps And Licensed By Type I Ifn, Diana Legarda, Scott J. Justus, Rosalind L. Ang, Nimisha Rikhi, Wenjing Li, Thomas M. Moran, Jianke Zhang, Emiko Mizoguchi, Matija Zelic, Michelle A. Kelliher, J. Magarian Blander, Adrian T. Ting

Open Access Articles

Tumor necrosis factor (TNF) induces necroptosis, a RIPK3/MLKL-dependent form of inflammatory cell death. In response to infection by Gram-negative bacteria, multiple receptors on macrophages, including TLR4, TNF, and type I IFN receptors, are concurrently activated, but it is unclear how they crosstalk to regulate necroptosis. We report that TLR4 activates CASPASE-8 to cleave and remove the deubiquitinase cylindromatosis (CYLD) in a TRIF- and RIPK1-dependent manner to disable necroptosis in macrophages. Inhibiting CASPASE-8 leads to CYLD-dependent necroptosis caused by the TNF produced in response to TLR4 ligation. While lipopolysaccharides (LPS)-induced necroptosis was abrogated in Tnf(-/-) macrophages, a soluble TNF ...


Er Stress In Temozolomide-Treated Glioblastomas Interferes With Dna Repair And Induces Apoptosis, Jessica L. Weatherbee, Jean-Louis Kraus, Alonzo H. Ross Jun 2016

Er Stress In Temozolomide-Treated Glioblastomas Interferes With Dna Repair And Induces Apoptosis, Jessica L. Weatherbee, Jean-Louis Kraus, Alonzo H. Ross

Open Access Articles

Glioblastoma multiforme (GBM) is a deadly grade IV brain tumor. Radiation in combination with temozolomide (TMZ), the current chemotherapeutic for GBMs, only provides 12-14 months survival post diagnosis. Because GBMs are dependent on both activation of the DNA damage pathway and the endoplasmic reticulum (ER) stress response, we asked if a novel ER stress inducing agent, JLK1486, increases the efficacy of TMZ. We found that the combination of TMZ+JLK1486 resulted in decreased proliferation in a panel of adherent GBM cells lines and reduced secondary sphere formation in non-adherent and primary lines. Decreased proliferation correlated with increased cell death due ...


The Secret Life Of Tethers: The Role Of Tethering Factors In Snare Complex Regulation, Michelle L. Dubuke, Mary Munson May 2016

The Secret Life Of Tethers: The Role Of Tethering Factors In Snare Complex Regulation, Michelle L. Dubuke, Mary Munson

Open Access Articles

Trafficking in eukaryotic cells is a tightly regulated process to ensure correct cargo delivery to the proper destination organelle or plasma membrane. In this review, we focus on how the vesicle fusion machinery, the SNARE complex, is regulated by the interplay of the multisubunit tethering complexes (MTC) with the SNAREs and Sec1/Munc18 (SM) proteins. Although these factors are used in different stages of membrane trafficking, e.g., Golgi to plasma membrane transport vs. vacuolar fusion, and in a variety of diverse eukaryotic cell types, many commonalities between their functions are being revealed. We explore the various protein-protein interactions and ...


Reciprocal Autoregulation By Nfi Occupancy And Etv1 Promotes The Developmental Expression Of Dendrite-Synapse Genes In Cerebellar Granule Neurons, Baojin Ding, John W. Cave, Paul R. Dobner, Debra Mullikin-Kilpatrick, Marina Bartzokis, Hong Zhu, Chi-Wing Chow, Richard M. Gronostajski, Daniel Lee Kilpatrick May 2016

Reciprocal Autoregulation By Nfi Occupancy And Etv1 Promotes The Developmental Expression Of Dendrite-Synapse Genes In Cerebellar Granule Neurons, Baojin Ding, John W. Cave, Paul R. Dobner, Debra Mullikin-Kilpatrick, Marina Bartzokis, Hong Zhu, Chi-Wing Chow, Richard M. Gronostajski, Daniel Lee Kilpatrick

Open Access Articles

Nuclear Factor One (NFI) transcription factors regulate temporal gene expression required for dendritogenesis and synaptogenesis via delayed occupancy of target promoters in developing cerebellar granule neurons (CGNs). Mechanisms that promote NFI temporal occupancy have not been previously defined. We show here that the transcription factor ETV1 directly binds to and is required for expression and NFI occupancy of a cohort of NFI-dependent genes in CGNs maturing in vivo. Expression of ETV1 is low in early postnatal cerebellum and increases with maturation, mirroring NFI temporal occupancy of coregulated target genes. Precocious expression of ETV1 in mouse CGNs accelerated onset of expression ...


Sumo-Targeted Ubiquitin Ligase (Stubl) Slx5 Regulates Proteolysis Of Centromeric Histone H3 Variant Cse4 And Prevents Its Mislocalization To Euchromatin, Kentaro Ohkuni, Yoshimitsu Takahashi, Alyona Fulp, Josh Lawrimore, Wei-Chun Au, Nagesh Pasupala, Reuben Levy-Myers, Jack Warren, Alexander Strunnikov, Richard E. Baker, Oliver Kerscher, Kerry Bloom, Munira A. Basrai May 2016

Sumo-Targeted Ubiquitin Ligase (Stubl) Slx5 Regulates Proteolysis Of Centromeric Histone H3 Variant Cse4 And Prevents Its Mislocalization To Euchromatin, Kentaro Ohkuni, Yoshimitsu Takahashi, Alyona Fulp, Josh Lawrimore, Wei-Chun Au, Nagesh Pasupala, Reuben Levy-Myers, Jack Warren, Alexander Strunnikov, Richard E. Baker, Oliver Kerscher, Kerry Bloom, Munira A. Basrai

Open Access Articles

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and ...


Regulation Of Chaperone Binding And Nucleosome Dynamics By Key Residues Within The Globular Domain Of Histone H3, Sarah J. Hainer, Joseph A. Martens Apr 2016

Regulation Of Chaperone Binding And Nucleosome Dynamics By Key Residues Within The Globular Domain Of Histone H3, Sarah J. Hainer, Joseph A. Martens

Open Access Articles

BACKGROUND: Nucleosomes have an important role in modulating access of DNA by regulatory factors. The role specific histone residues have in this process has been shown to be an important mechanism of transcription regulation. Previously, we identified eight amino acids in histones H3 and H4 that are required for nucleosome occupancy over highly transcribed regions of the genome.

RESULTS: We investigate the mechanism through which three of these previously identified histone H3 amino acids regulate nucleosome architecture. We find that histone H3 K122, Q120, and R49 are required for Spt2, Spt6, and Spt16 occupancies at genomic locations where transcription rates ...


Neat1 Is Required For Survival Of Breast Cancer Cells Through Fus And Mir-548, Hao Ke, Limin Zhao, Xu Feng, Haibo Xu, Li Zou, Qin Yang, Xiaosan Su, Lingtao Peng, Baowei Jiao Apr 2016

Neat1 Is Required For Survival Of Breast Cancer Cells Through Fus And Mir-548, Hao Ke, Limin Zhao, Xu Feng, Haibo Xu, Li Zou, Qin Yang, Xiaosan Su, Lingtao Peng, Baowei Jiao

Open Access Articles

Increasing evidence shows that long noncoding RNAs (lncRNAs) have important roles in the regulation of multiple cellular processes, including cell division, cell growth, and apoptosis, as well as cancer metastasis and neurological disease progression; however, the mechanism of how lncRNAs regulate these processes is not well established. In this study, we demonstrated that downregulating the expression of the lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in breast cancer cells inhibited cell growth and induced cell apoptosis. In addition, the RNA-binding protein fused in sarcoma/translocated in liposarcoma (FUS/TLS) physically interacted with NEAT1, and reducing the expression of FUS/TLS ...