DOCSLIB.ORG

Nuclear Non-Chromatin Proteinaceous Structures: Their Role in the Organization and Function of the Interphase Nucleus

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

J. Cell Set. 44, 395-435 (1980) 295 Printed in Great Britain © Company of BiologitU Limited igSo NUCLEAR NON-CHROMATIN PROTEINACEOUS STRUCTURES: THEIR ROLE IN THE ORGANIZATION AND FUNCTION OF THE INTERPHASE NUCLEUS PAUL S. AGUTTER* AND JONATHAN C. W. RICHARDSONf • Department of Biological Sciences, Napier College, Colinton Road, Edinburgh EH10 5DT, Scotland and f Department of Physiology and Pharmacology, University of St Andrews, Bute Medical Buildings, St Andrews, Fife, Scotland REVIEW ARTICLE: CONTENTS I. INTRODUCTION page 39s (1) Historical background 395 (2) Nomenclature 397 II. NUCLEAR PROTEIN MATRIX AND NUCLEAR GHOSTS 397 (1) Isolation 397 (2) Composition 398 (3) Ultrastructure 401 (4) Enzyme activities associated with the nuclear protein matrix 405 (5) Contractility of the nuclear protein matrix 405 (6) Functions associated with the nucleai protein matrix 408 III. SUBFRACTIONS OF THE NUCLEAR PROTEIN MATRIX 411 (A) The pore-lamina 411 (1) Isolation 411 (2) Composition 413 (3) Ultrastructure 414 (4) The molecular organization of the pore-lamina 417 (B) Other subfractions 419 IV. COMPOSITIONAL AND FUNCTIONAL DIFFERENCES BETWEEN THE PORE-LAMINA AND THE REMAINDER OF THE NUCLEAR PROTEIN MATRIX 42O V. PROSPECTS FOR FURTHER RESEARCH 422 (1) The role of the nuclear protein matrix in nucleo-cytoplasmic RNA transport 422 (2) Relevance of a knowledge of factors affecting the stability of the intra- nuclear regions of the matrix to the further development of methods for isolation of the nuclear envelope 423 (3) Fate of the nuclear matrix during mitosis 423 I. INTRODUCTION (1) Historical background Since 1949 there have been several accounts of a 'honeycomb layer' or 'nuclear cortex' in the nuclei of lower eukaryotes (Callan, Randall & Tomlin, 1949; Callan & Tomlin, 1950; Harris & James, 1952; Pappas, 1956; Beams, Tahmisian, Devine & 26-2 396 P. S. Agutter andj. C. W. Richardson Anderson, 1957; Mercer, 1959; Gray & Guillery, 1963; Daniels & Breyer, 1967; Barton, Kisieleski, Wassermann & Mackevicius, 1971). This highly structured layer appears either to separate the inner nuclear membrane from the peripheral chromatin or to structure the heterochromatin region. It extends for up to 300 nm into the nucleoplasm, though this distance varies considerably with cell type, and shows discontinuities in the region of the pore complexes. In the decade 1960-70 a similar, but less extensive highly organized layer of material was described in the nuclei of higher eukaryotes. The structure was termed, alternatively, the 'granular perinuclear layer' (Bruni & Porter, 1965), 'dense lamella' (Kalifat, Bouteille & Delarue, 1967), 'fibrous lamina' (Coggeshall & Fawcett, 1964; Fawcett, 1966; Ghadially, Bhatnager & Fuller, 1972; Cohen & Sundeen, 1976) or 'zonula nucleum limitans' (Mazanek, 1967; Patrizi & Poger, 1967). Davies and his co-workers, in a detailed study of this layer, found that it comprised alternating narrow electron-dense and electron-transparent bands arranged parallel to the envelope, intersected orthogonally by fine electron-dense fibrils (Davies, 1967, 1968; Davies & Small, 1968). Detailed investigation of amphibian oocyte nuclear envelopes revealed, instead of this perinuclear layer, an array of fibres extending from the nuclear face of the pore complexes into the nucleoplasm (Franke & Scheer, 1970). Collectively, these studies suggested that at least some part of the region of the nucleus contiguous with the inner nuclear membrane is structured by a network of fibrils continuous with the pore complexes. Such a structure has since been isolated and termed the 'nuclear pore-lamina fraction' (Aaronson & Blobel, 1974, 1975). Over the years 1942-69, evidence accrued for the existence of a structural network of non-chromatin protein fibrils extending throughout the nucleus (Mayer & Gulick, 1942; Zbarsky & Debov, 1948; Wang et al. 1950; Allfrey, Dally & Mirsky, 1955; Du Praw, 1956; Zbarsky & Georgiev, 1959; Georgiev & Chentsov, 1962; Zbarsky, Dmitrieva & Yermolayeva, 1962; Wang, 1961, 1966; Steel&Busch, 1963; Bernhard & Granbonlan, 1963; Swift, 1963; Holtzman, Smith & Penman, 1966; Kaye & McMaster-Kaye, 1966; Monneron & Bernhard, 1969). Such a network has since been isolated (Berezney & Coffey, 1974a, b) and termed the 'nuclear protein matrix'. The early evidence for the structure depended on its resistance to the procedures used for the solubilization of chromatin: it retained the original shape of the nucleus after extraction with 2 M NaCl and dilute alkali. Corroborating evidence included: (a) the observation that while nuclease treatment did not markedly alter the shape and size of the nucleus, protease treatment quickly resulted in swelling and rupture (Anderson, 1953); and (b) the maintenance of the gross morphology of the nucleus after removal of the nuclear membranes with non-ionic detergents (Bach & Johnston, 1967). Later investigations by Busch and co-workers led to the idea of a structural ribonucleo- protein network, rather than a merely proteinaceous structure, extending throughout the nucleus (Smetana, Steele & Busch, 1963; Steele & Busch, 1966; Narayan, Steele, Smetana & Busch, 1967). Before 1970, therefore, there was a substantial body of literature concerning at least 2 sorts of non-membranous structural elements inside nuclei. It seems reasonable a priori to regard the juxtamembranous layer described in detail by Davies (1968) as Nuclear matrix, ghost and pore-lamina 397 the peripheral portion of the protein or ribonucleoprotein network discussed above. Since 1970, several biochemical studies of these structural elements have been carried out. The present review is devoted to the implications of these studies for an under- standing of the organization of nuclei and their functions during interphase. (2) Nomenclature Before discussion of these studies can be undertaken, clarification of the terminology is essential. Different laboratories naturally develop different systems of nomenclature; the system outlined here is based, as far as possible, on the most frequent current usage. It should be emphasized that our terminology does not distinguish between a structure observed in situ by microscopy and an apparently similar but not necessarily identical structure prepared by subfractionation of isolated nuclei. To make such a distinction would greatly complicate the nomenclature, but its importance in the interpretation of experimental findings should not be overlooked. We use the term nuclear envelope to describe the peripheral structure isolated from nuclei. The nuclear envelope has 4 ultrastructurally distinct subfractions: outer nuclear membrane, inner nuclear membrane, pore complexes and fibrous lamina. Removal of the 2 membranes leaves the ' nuclear pore-lamina fraction' or, more briefly, pore-lamina (see above and section in A, below). Careful removal of the chromatin and the 2 nuclear membranes from whole nuclei leaves a structure which has been called the nuclear protein matrix (see above and section 11, below). This structure includes the pore-lamina: pore complexes inter- connected with fibrils are visible in it. If nuclei are vigorously extracted with deter- gent and high-ionic-strength media but not treated with nucleases, nuclear ghosts can be banded out on sucrose gradients. The distinction between these nuclear ghosts (which are operationally distinct from nuclear envelopes) and the nuclear protein matrix is discussed in section 11. The nuclear protein matrix appears to contain residual nucleoli as morphologically distinct regions. II. NUCLEAR PROTEIN MATRICES AND NUCLEAR GHOSTS (1) Isolation The nuclear protein matrix. Berezney, Coffey and co-workers have evolved a pro- cedure for isolating nuclear protein matrix from whole nuclei (Berezney & Coffey 1974a, b, 1975a, b, 1977). Essentially, the method involves: (a) swelling of the nuclei (isolated by a variant of the method of Blobel & Potter, 1966) by overnight storage at 5 °C and treatment with a buffer containing a low concentration (0-2 ITIM) of mag- nesium ions; (b) solubilization of the chromatin in 2 M NaCl; (c) removal of the nuclear membranes with 1 % (w/v) Triton X-100; and (d) removal of residual nucleic acids with DNase and RNase. More recently, a modified procedure involving omission of step (a) and inclusion of inhibitors of proteolysis in the media has been employed. This modified procedure has been claimed to result in the production of more intact matrices (Berezney & Buchholtz, 1978). This or closely related methods for isolating nuclear protein matrices have been successfully used in other laboratories (Shelton, 398 P. S. Agutter andj. C. W. Richardson Cobbs, Povlishock & Burkat, 1976; Faiferman & Pogo, 1975; Hildebrand, Okinaka & Gurley, 1975; Hodge, Mancini, Davis & Heywood, 1977; Agutter & Birchall, 1979). Faiferman & Pogo (1975) have also isolated the matrix fraction, albeit in a fragmented state, from nuclei disrupted by nitrogen cavitation or in a French pressure cell. The nuclear ghost. Riley, Keller and co-workers have described the isolation of nuclear ghosts by a procedure involving: (a) washing of the nuclei in 1 % Tween 40 and 0-5% (w/v) sodium deoxycholate; (b) disruption of the nuclear contents with 0-5 M MgCl2; and (c) sucrose gradient centrifugation. The nuclear ghosts band on the gradient at 47-52% (w/v) sucrose (Riley, Keller & Byers, 1975; Riley & Keller, 1976a, b, 1978a, b; Keller & Riley, 19760, b). (2) Composition The nuclear protein matrix. The nuclear protein matrix accounts for about 10-20% of the total nuclear
Recommended publications
  • Dynamic Force-Induced Direct Dissociation of Protein Complexes in a Nuclear Body in Living Cells

    Dynamic Force-Induced Direct Dissociation of Protein Complexes in a Nuclear Body in Living Cells

    ARTICLE Received 13 Jan 2012 | Accepted 26 Apr 2012 | Published 29 May 2012 DOI: 10.1038/ncomms1873 Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells Yeh-Chuin Poh1, Sergey P. Shevtsov2, Farhan Chowdhury1, Douglas C. Wu1, Sungsoo Na3, Miroslav Dundr2 & Ning Wang1 Despite past progress in understanding mechanisms of cellular mechanotransduction, it is unclear whether a local surface force can directly alter nuclear functions without intermediate biochemical cascades. Here we show that a local dynamic force via integrins results in direct displacements of coilin and SMN proteins in Cajal bodies and direct dissociation of coilin-SMN associated complexes. Spontaneous movements of coilin increase more than those of SMN in the same Cajal body after dynamic force application. Fluorescence resonance energy transfer changes of coilin-SMN depend on force magnitude, an intact F-actin, cytoskeletal tension, Lamin A/C, or substrate rigidity. Other protein pairs in Cajal bodies exhibit different magnitudes of fluorescence resonance energy transfer. Dynamic cyclic force induces tiny phase lags between various protein pairs in Cajal bodies, suggesting viscoelastic interactions between them. These findings demonstrate that dynamic force-induced direct structural changes of protein complexes in Cajal bodies may represent a unique mechanism of mechanotransduction that impacts on nuclear functions involved in gene expression. 1 Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Champaign, Illinois 61801, USA. 2 Department of Cell Biology, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064, USA. 3 Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indiana 46202, USA.
  • 1 the Nucleoporin ELYS Regulates Nuclear Size by Controlling NPC

    1 the Nucleoporin ELYS Regulates Nuclear Size by Controlling NPC

    bioRxiv preprint doi: https://doi.org/10.1101/510230; this version posted January 2, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity Predrag Jevtić1,4, Andria C. Schibler2,4, Gianluca Pegoraro3, Tom Misteli2,*, Daniel L. Levy1,* 1 Department of Molecular Biology, University of Wyoming, Laramie, WY, 82071 2 National Cancer Institute, NIH, Bethesda, MD, 20892 3 High Throughput Imaging Facility (HiTIF), National Cancer Institute, NIH, Bethesda, MD, 20892 4 Co-first authors *Corresponding authors: Daniel L. Levy Tom Misteli University of Wyoming National Cancer Institute, NIH Department of Molecular Biology Center for Cancer Research 1000 E. University Avenue 41 Library Drive, Bldg. 41, B610 Laramie, WY, 82071 Bethesda, MD, 20892 Phone: 307-766-4806 Phone: 240-760-6669 Fax: 307-766-5098 Fax: 301-496-4951 E-mail: [email protected] E-mail: [email protected] Running Head: ELYS is a nuclear size effector Abbreviations: NE, nuclear envelope; NPC, nuclear pore complex; ER, endoplasmic reticulum; Nup, nucleoporin; FG-Nup, phenylalanine-glycine repeat nucleoporin 1 bioRxiv preprint doi: https://doi.org/10.1101/510230; this version posted January 2, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
  • Effects of Hyperthermia on Chromatin Condensation and Nucleoli

    Effects of Hyperthermia on Chromatin Condensation and Nucleoli

    (CANCER RESEARCH 49, 1254-1260. March 1. 1989] Effects of Hyperthermia on Chromatin Condensation and Nucleoli Disintegration as Visualized by Induction of Premature Chromosome Condensation in Interphase Mammalian Cells1 George E. Iliakis and Gabriel E. Pantelias2 Thomas Jefferson University Hospital, Department of Radiation Oncology and Nuclear Medicine, Philadelphia, Pennsylvania 19107 [G. E. I., G. E. P.]; and the National Research Center for Physical Sciences "Demokritos", Aghia Paraskevi Attikis, Athens, Greece [G. E. P.] ABSTRACT nuclei (13), in chromatin (14-17), and in nuclear matrices (18, 19), and it was proposed that disruption of important nuclear The effects of hyperthermia on chromatin condensation and nucleoli processes by this nuclear protein binding may be the reason for disintegration, as visualized by induction of premature chromosome con cell killing (17). Beyond cell killing the excess nuclear proteins densation in interphase mammalian cells, was studied in exponentially have been implicated in the inhibition of DNA synthesis (5, 6) growing and plateau phase Chinese hamster ovary cells. Exposure to heat reduced the ability of interphase chromatin to condense and the and the inhibition of DNA repair following both ionizing (20, ability of the nucleolar organizing region to disintegrate under the influ 21) and uv (22) irradiation. It is thought that inhibition of ence of factors provided by mitotic cells when fused to interphase cells. these cellular functions may be due to alterations induced in Based on these effects treated cells were classified in three categories. chromatin conformation and in particular to restriction of DNA Category 1 contained cells able to condense their chromatin and disinte supercoiling changes as a result of protein addition to the grate the nucleolar organizing region.
  • Snapshot: Cellular Bodies David L

    Snapshot: Cellular Bodies David L

    SnapShot: Cellular Bodies David L. Spector Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA Number/ Typical Size Marker Body Name Description Image Cell and Shape Protein Involved in snRNP and snoRNP biogenesis and 0.1–2.0 µm; Cajal Body 0–6 Coilin posttranscriptional modification of newly assembled round spliceosomal snRNAs. 20S core Contains ubiquitin conjugates, the proteolytically active 0.2–1.2 µm; catalytic Clastosome 0–3 20S core and 19S regulatory complexes of the 26S irregular component of proteasome, and protein substrates of the proteasome. proteasome Contains several factors involved in 3′ cleavage of mRNAs. 0.2–1.0 µm; Cleavage Body 1–4 CstF 64 kDa ?20% contain newly synthesized RNA. Some cleavage round bodies localize adjacent to Cajal and PML bodies. Nuclear Contains proteins for pre-mRNA processing. Involved in Speckle or 0.8–1.8 µm; SC35, 25–50 the storage, assembly, and/or modification of pre-mRNA Interchromatin irregular SF2/ASF splicing factors. Granule Cluster Induced by heat shock response. Associates with Nuclear Stress 0.3–3.0 µm; satellite III repeats on human chromosome 9q12 and 2–10 HSF1 Body irregular other pericentromeric regions; recruits various RNA- binding proteins. Contains several transcription factors (Oct1/PTF) and 1.0–1.5 µm; OPT Domain 1–3 PTF RNA transcripts; predominant in late G1 cells. Often round Nuclear Bodies localizes close to nucleolus. 0.5 µm; Contains several RNA-binding proteins and nuclear- Paraspeckle 10–20 p54nrb, PSP1 round retained CTN-RNA. Cap on surface of nucleolus; found mainly in transformed Perinucleolar 0.3–1.0 µm; 1–4 hnRNPI (PTB) cells.
  • Paraspeckles: Possible Nuclear Hubs by the RNA for the RNA

    Paraspeckles: Possible Nuclear Hubs by the RNA for the RNA

    BioMol Concepts, Vol. 3 (2012), pp. 415–428 • Copyright © by Walter de Gruyter • Berlin • Boston. DOI 10.1515/bmc-2012-0017 Review Paraspeckles: possible nuclear hubs by the RNA for the RNA Tetsuro Hirose 1, * and Shinichi Nakagawa 2 Introduction 1 Biomedicinal Information Research Center , National Institute of Advanced Industrial Science and Technology, The eukaryotic cell nucleus is highly compartmentalized. 2-4-7 Aomi, Koutou 135-0064, Tokyo , Japan More than 10 membraneless subnuclear organelles have 2 RNA Biology Laboratory , RIKEN Advanced Research been identifi ed (1, 2) . These so-called nuclear bodies exist Institute, 2-1 Hirosawa, Wako 351-0198 , Japan in the interchromosomal space, where they are enriched in multiple nuclear regulatory factors, such as transcription and * Corresponding author RNA-processing factors. These factors are thought to serve e-mail: [email protected] as specialized hubs for various nuclear events, including transcriptional regulation and RNA processing (3, 4) . Some nuclear bodies serve as sites for the biogenesis of macromo- Abstract lecular machineries, such as ribosomes and spliceosomes. Multiple cancer cell types show striking alterations in their The mammalian cell nucleus is a highly compartmental- nuclear body organization, including changes in the numbers, ized system in which multiple subnuclear structures, called shapes and sizes of certain nuclear bodies (5) . The structural nuclear bodies, exist in the nucleoplasmic spaces. Some of complexity and dynamics of nuclear bodies have been impli- the nuclear bodies contain specifi c long non-coding RNAs cated in the regulation of complex gene expression pathways (ncRNAs) as their components, and may serve as sites for in mammalian cells.
  • The Kinesin Spindle Protein Inhibitor Filanesib Enhances the Activity of Pomalidomide and Dexamethasone in Multiple Myeloma

    The Kinesin Spindle Protein Inhibitor Filanesib Enhances the Activity of Pomalidomide and Dexamethasone in Multiple Myeloma

    Plasma Cell Disorders SUPPLEMENTARY APPENDIX The kinesin spindle protein inhibitor filanesib enhances the activity of pomalidomide and dexamethasone in multiple myeloma Susana Hernández-García, 1 Laura San-Segundo, 1 Lorena González-Méndez, 1 Luis A. Corchete, 1 Irena Misiewicz- Krzeminska, 1,2 Montserrat Martín-Sánchez, 1 Ana-Alicia López-Iglesias, 1 Esperanza Macarena Algarín, 1 Pedro Mogollón, 1 Andrea Díaz-Tejedor, 1 Teresa Paíno, 1 Brian Tunquist, 3 María-Victoria Mateos, 1 Norma C Gutiérrez, 1 Elena Díaz- Rodriguez, 1 Mercedes Garayoa 1* and Enrique M Ocio 1* 1Centro Investigación del Cáncer-IBMCC (CSIC-USAL) and Hospital Universitario-IBSAL, Salamanca, Spain; 2National Medicines Insti - tute, Warsaw, Poland and 3Array BioPharma, Boulder, Colorado, USA *MG and EMO contributed equally to this work ©2017 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol. 2017.168666 Received: March 13, 2017. Accepted: August 29, 2017. Pre-published: August 31, 2017. Correspondence: [email protected] MATERIAL AND METHODS Reagents and drugs. Filanesib (F) was provided by Array BioPharma Inc. (Boulder, CO, USA). Thalidomide (T), lenalidomide (L) and pomalidomide (P) were purchased from Selleckchem (Houston, TX, USA), dexamethasone (D) from Sigma-Aldrich (St Louis, MO, USA) and bortezomib from LC Laboratories (Woburn, MA, USA). Generic chemicals were acquired from Sigma Chemical Co., Roche Biochemicals (Mannheim, Germany), Merck & Co., Inc. (Darmstadt, Germany). MM cell lines, patient samples and cultures. Origin, authentication and in vitro growth conditions of human MM cell lines have already been characterized (17, 18). The study of drug activity in the presence of IL-6, IGF-1 or in co-culture with primary bone marrow mesenchymal stromal cells (BMSCs) or the human mesenchymal stromal cell line (hMSC–TERT) was performed as described previously (19, 20).
  • Quantification of Nuclear Protein Dynamics Reveals Chromatin Remodeling During Acute Protein Degradation

    Quantification of Nuclear Protein Dynamics Reveals Chromatin Remodeling During Acute Protein Degradation

    bioRxiv preprint doi: https://doi.org/10.1101/345686; this version posted June 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Quantification of nuclear protein dynamics reveals chromatin remodeling during acute protein degradation Alexander J. Federation1, Vivek Nandakumar1, Hao Wang1, Brian Searle2, Lindsay Pino2, Gennifer Merrihew2, Sonia Ting2, Nicholas Howard1, Tanya Kutyavin1, Michael J. MacCoss2, John A. Stamatoyannopoulos1,2 1. Altius Institute for Biomedical Sciences; Seattle, WA 98121 2. University of Washington, Department of Genome Sciences; Seattle, WA 98195 Correspondence: John A. Stamatoyannopoulos [email protected] Michael J. MacCoss [email protected] bioRxiv preprint doi: https://doi.org/10.1101/345686; this version posted June 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Sequencing-based technologies cannot measure post-transcriptional dynamics of the nuclear proteome, but unbiased mass-spectrometry measurements of nuclear proteins remain difficult. In this work, we have combined facile nuclear sub-fractionation approaches with data-independent acquisition mass spectrometry to improve detection and quantification of nuclear proteins in human cells and tissues. Nuclei are isolated and subjected to a series of extraction conditions that enrich for nucleoplasm, euchromatin, heterochromatin and nuclear-membrane associated proteins. Using this approach, we can measure peptides from over 70% of the expressed nuclear proteome. As we are physically separating chromatin compartments prior to analysis, proteins can be assigned into functional chromatin environments that illuminate systems-wide nuclear protein dynamics.
  • Human Autoantibody to a Novel Protein of the Nuclear Coiled Body: Immunological Characterization and Cdna Cloning of P80-Coilin by Luis E

    Human Autoantibody to a Novel Protein of the Nuclear Coiled Body: Immunological Characterization and Cdna Cloning of P80-Coilin by Luis E

    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Human Autoantibody to a Novel Protein of the Nuclear Coiled Body: Immunological Characterization and cDNA Cloning of p80-coilin By Luis E. C. Andrade, Edward K. L. Chan, Ivan Raska, Carol L. Peebles, Goran Roos, and Eng M . Tan From the W. M. Keck Autoimmune Disease Center, Department ofMolecular and Experimental Medicine, Scripps Clinic and Research Foundation, La Jolla, California 92037 Summary Antibodies producing an unusual immunofluorescent pattern were identified in the sera ofpatients with diverse autoimmune features. This pattern was characterized by the presence of up to six round discrete nuclear bodies in interphase cell nuclei. Immunoblotting analysis showed that these sera recognized an 80-kD nuclear protein, and affinity-purified anti-p80 antibody from the protein band reproduced the fluorescent staining of nuclear bodies. Colloidal gold immunoelectron microscopy showed that the affinity-purified anti-p80 antibody recognized the coiled body, an ultramicroscopic nuclear structure probably first described by the Spanish cytologist Ramon y Cajal. Five cDNA clones were isolated from a MOLT-4 cell Xgt-11 expression library using human antibody and oligonucleotide probes. The longest cDNA insert was 2.1 kb and had an open reading frame of 405 amino acids. A clone encoding a 14-kD COOH-terminal region of the protein was used for expression of a O-galactosidase fusion protein . An epitope was present in this COOH-terminal 14-kD region, which was recognized by 18 of 20 sera with anti-p80 reactivity, and affinity-purified antibody from the recombinant protein also reacted in immunofluorescence to show specific staining of the coiled body.
  • Nucleocytoplasmic Transport: Regulatory Mechanisms and the Implications in Neurodegeneration

    Nucleocytoplasmic Transport: Regulatory Mechanisms and the Implications in Neurodegeneration

    International Journal of Molecular Sciences Review Nucleocytoplasmic Transport: Regulatory Mechanisms and the Implications in Neurodegeneration Baojin Ding * and Masood Sepehrimanesh Department of Biology, University of Louisiana at Lafayette, 410 East Saint Mary Boulevard, Lafayette, LA 70503, USA; [email protected] * Correspondence: [email protected] Abstract: Nucleocytoplasmic transport (NCT) across the nuclear envelope is precisely regulated in eukaryotic cells, and it plays critical roles in maintenance of cellular homeostasis. Accumulating evidence has demonstrated that dysregulations of NCT are implicated in aging and age-related neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer’s disease (AD), and Huntington disease (HD). This is an emerging research field. The molecular mechanisms underlying impaired NCT and the pathogenesis leading to neurodegener- ation are not clear. In this review, we comprehensively described the components of NCT machinery, including nuclear envelope (NE), nuclear pore complex (NPC), importins and exportins, RanGTPase and its regulators, and the regulatory mechanisms of nuclear transport of both protein and transcript cargos. Additionally, we discussed the possible molecular mechanisms of impaired NCT underlying aging and neurodegenerative diseases, such as ALS/FTD, HD, and AD. Keywords: Alzheimer’s disease; amyotrophic lateral sclerosis; Huntington disease; neurodegenera- tive diseases; nuclear pore complex; nucleocytoplasmic transport; Ran GTPase Citation: Ding, B.; Sepehrimanesh, M. Nucleocytoplasmic Transport: Regulatory Mechanisms and the Implications in Neurodegeneration. 1. Introduction Int. J. Mol. Sci. 2021, 22, 4165. As a hallmark of eukaryotic cells, the genetic materials are separated from the cyto- https://doi.org/10.3390/ijms plasmic contents by a highly regulated membrane, called nuclear envelope (NE), which 22084165 has two concentric bilayer membranes, the inner nuclear membrane (INM), and outer nuclear membrane (ONM).
  • Exploring Mammalian Genome Within Phase-Separated Nuclear Bodies: Experimental Methods and Implications for Gene Expression

    Exploring Mammalian Genome Within Phase-Separated Nuclear Bodies: Experimental Methods and Implications for Gene Expression

    G C A T T A C G G C A T genes Review Exploring Mammalian Genome within Phase-Separated Nuclear Bodies: Experimental Methods and Implications for Gene Expression Annick Lesne 1,2,*, Marie-Odile Baudement 1,3 , Cosette Rebouissou 1 and Thierry Forné 1,* 1 IGMM, Univ. Montpellier, CNRS, F-34293 Montpellier, France; [email protected] (M.-O.B.); [email protected] (C.R.) 2 Sorbonne Université, CNRS, Laboratoire de Physique Théorique de la Matière Condensée, LPTMC, F-75252 Paris, France 3 Centre for Integrative Genetics (CIGENE), Faculty of Biosciences, Norwegian University of Life Sciences, 1430 Ås, Norway * Correspondence: [email protected] (A.L.); [email protected] (T.F.); Tel.: +33-434-359-682 (T.F.) Received: 6 November 2019; Accepted: 13 December 2019; Published: 17 December 2019 Abstract: The importance of genome organization at the supranucleosomal scale in the control of gene expression is increasingly recognized today. In mammals, Topologically Associating Domains (TADs) and the active/inactive chromosomal compartments are two of the main nuclear structures that contribute to this organization level. However, recent works reviewed here indicate that, at specific loci, chromatin interactions with nuclear bodies could also be crucial to regulate genome functions, in particular transcription. They moreover suggest that these nuclear bodies are membrane-less organelles dynamically self-assembled and disassembled through mechanisms of phase separation. We have recently developed a novel genome-wide experimental method, High-salt Recovered Sequences sequencing (HRS-seq), which allows the identification of chromatin regions associated with large ribonucleoprotein (RNP) complexes and nuclear bodies.
  • Cell Cycle Arrest Through Indirect Transcriptional Repression by P53: I Have a DREAM

    Cell Cycle Arrest Through Indirect Transcriptional Repression by P53: I Have a DREAM

    Cell Death and Differentiation (2018) 25, 114–132 Official journal of the Cell Death Differentiation Association OPEN www.nature.com/cdd Review Cell cycle arrest through indirect transcriptional repression by p53: I have a DREAM Kurt Engeland1 Activation of the p53 tumor suppressor can lead to cell cycle arrest. The key mechanism of p53-mediated arrest is transcriptional downregulation of many cell cycle genes. In recent years it has become evident that p53-dependent repression is controlled by the p53–p21–DREAM–E2F/CHR pathway (p53–DREAM pathway). DREAM is a transcriptional repressor that binds to E2F or CHR promoter sites. Gene regulation and deregulation by DREAM shares many mechanistic characteristics with the retinoblastoma pRB tumor suppressor that acts through E2F elements. However, because of its binding to E2F and CHR elements, DREAM regulates a larger set of target genes leading to regulatory functions distinct from pRB/E2F. The p53–DREAM pathway controls more than 250 mostly cell cycle-associated genes. The functional spectrum of these pathway targets spans from the G1 phase to the end of mitosis. Consequently, through downregulating the expression of gene products which are essential for progression through the cell cycle, the p53–DREAM pathway participates in the control of all checkpoints from DNA synthesis to cytokinesis including G1/S, G2/M and spindle assembly checkpoints. Therefore, defects in the p53–DREAM pathway contribute to a general loss of checkpoint control. Furthermore, deregulation of DREAM target genes promotes chromosomal instability and aneuploidy of cancer cells. Also, DREAM regulation is abrogated by the human papilloma virus HPV E7 protein linking the p53–DREAM pathway to carcinogenesis by HPV.Another feature of the pathway is that it downregulates many genes involved in DNA repair and telomere maintenance as well as Fanconi anemia.
  • The Role of Nuclear Envelope Proteins in Chromatin Organization, Differentiation and Disease Distinct Degenerative Disorders, Referred to As Laminopathies

    The Role of Nuclear Envelope Proteins in Chromatin Organization, Differentiation and Disease Distinct Degenerative Disorders, Referred to As Laminopathies

    Cecilia Bergqvist The genetic material is highly structured within the nucleus, with transcriptionally inactive heterochromatin enriched at the nuclear The role of nuclear envelope periphery and active euchromatin in the nuclear interior. The nuclear lamina together with several hundred nuclear envelope transmembrane proteins in chromatin proteins (NETs) connect chromatin to the nuclear periphery. Most NETs are tissue-specific and uncharacterized, with mutations linked to organization, differentiation and The role of nuclear envelope proteins in chromatin organization, differentiation and disease differentiation organization, in chromatin proteins of nuclear envelope role The distinct degenerative disorders, referred to as laminopathies. The NET disease primarily studied in this thesis is called Spindle-Associated Membrane Protein 1 (Samp1). We showed that overexpression of Samp1 induced a fast differentiation of human induced pluripotent stem cells and that the binding between two NETs, Samp1 and Emerin, is regulated by Cecilia Bergqvist RanGTP. Another focus of this thesis was the development of a novel method, Fluorescent Ratiometric Imaging of Chromatin (FRIC). FRIC quantitatively monitors the epigenetic state of chromatin in live cells. Using FRIC, we were able to show that Samp1 promotes peripheral heterochromatin organization. FRIC also detected an increased distribution of heterochromatin at the nuclear periphery during neuronal differentiation. In conclusion, FRIC is a useful tool that could serve medical research in elucidating