DOCSLIB.ORG

CENTROSOME NUMBER HOMEOSTASIS: LESSONS from CEP135 ISOFORM DYSREGULATION in BREAST CANCER by DIVYA GANAPATHI SANKARAN Bachelor

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
CENTROSOME NUMBER HOMEOSTASIS: LESSONS from CEP135 ISOFORM DYSREGULATION in BREAST CANCER by DIVYA GANAPATHI SANKARAN Bachelor CENTROSOME NUMBER HOMEOSTASIS: LESSONS FROM CEP135 ISOFORM DYSREGULATION IN BREAST CANCER By DIVYA GANAPATHI SANKARAN Bachelor of Technology, Anna University, India, 2013 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Cancer Biology Program 2019 This thesis for the Doctor of Philosophy degree by Divya Ganapathi Sankaran has been approved for the Cancer Biology Program By Rytis Prekeris, Chair Jeffrey Moore David Bentley Heide Ford Mary Reyland Chad G Pearson, Advisor Date: 05/17/2019 ii Ganapathi Sankaran, Divya (Ph.D., Cancer Biology) Centrosome Number Homeostasis: Lessons from CEP135 Isoform Dysregulation in Breast Cancer Thesis Directed by Associate Professor Chad G. Pearson ABSTRACT The centrosome, comprised of two centrioles surrounded by pericentriolar material, is the cell’s central microtubule organizing center. Centrosome duplication is coupled with the cell cycle such that centrosomes duplicate once in S phase. Loss of such coupling produces supernumerary centrosomes, a condition called centrosome amplification (CA). CA can promote hallmarks of tumorigenesis. In this thesis, I investigate the contribution of centriole overduplication to CA and its consequences on microtubule organization and genomic stability in breast cancer cells. CEP135, a centriole assembly protein, is dysregulated in some breast cancers. We previously identified a short isoform of CEP135, CEP135mini that represses centriole duplication. CEP135mini represses centriole duplication by limiting the localization of essential proteins required for centriole duplication. Interestingly, the relative level of CEP135full to CEP135mini (the CEP135full:mini ratio) is higher in centrosome amplified breast cancer cell lines. Specifically, I demonstrate that inducing expression of CEP135full increases CA. In contrast, elevating CEP135mini reduces centrosome number in breast cancer cells. I find that the CEP135 isoforms in vivo are generated by alternative polyadenylation. A directed genetic mutation near the CEP135mini alternative polyadenylation signal reduces the CEP135full:mini ratio and decreases CA, accordingly. Thus, dysregulation of the CEP135 isoforms can promote CA in breast cancer cells. Furthermore, in order to iii characterize the consequences of CA in microtubule organization and chromosome segregation, we designed a semi-automatic algorithm using machine learning to detect and analyze centrosomes and microtubule organization. Using this algorithm, I find that centrosome amplified breast cancer cells have microtubule organization defects such as increased microtubule density. Moreover, dysregulation of CEP135 isoforms that promotes CA also increases the frequency of multipolar spindles, anaphase-lagging chromosomes, and micronuclei leading to chromosome segregation defects. I conclude that dysregulation of the CEP135 isoforms can promote centriole overduplication and results in chromosome segregation errors in breast cancer cells. The form and content of this abstract are approved. I recommend its publication. Approved: Chad G Pearson iv ACKNOWLEDGEMENTS I am very grateful to Dr. Chad G Pearson for all the life lessons I have acquired from him over the past five years. He has taught me to be focused, to be thoughtful and to never stop questioning. Chad has shown respect for me as a scientist through his personal investment in my learning. I am very thankful for all his hard work and have enjoyed being his student. I especially would like to thank him for giving me those chances to bounce back when I struggled and for space he provided me to dig intellectually deeper, grow and be successful. I would also like to thank my lab members Alexander Stemm-Wolf, Kristin Dahl, Dr. Nick Galati, Dr. Marisa Ruehle, Dr. Brian Bayless, Anthony Junker, and Adam Soh for creating a very collaborative and friendly environment for the last five years. My lab members have believed in me and encouraged me on my hardest days. I have learned many things from all my lab members and they have been my home away from India. I would like to dedicate my thesis to my husband Dr. Bharath Hariharan, my parents Ganapathi Sankaran, Banumathi Ganapathi Sankaran and, my sister Dr. Jayadurga Ganapathi Sankaran. My parents have worked very hard and selflessly for me. Bharath has motivated me relentlessly. They showed me how to work hard, taught me to never look down upon failures and be very courageous throughout this journey. I would also like to acknowledge my-in-laws Rama Hariharan and Hariharan, who have been very kind and supported me throughout my graduate career. Most importantly, I would also like to thank my committee members Dr. Jeffery Moore, Dr. Rytis Prekeris, Dr. David Bentley, Dr. Mary Reyland and Dr. Heide Ford for all the great advice and the Cancer Biology Program for helping me pursue my interests. v TABLE OF CONTENTS CHAPTER CENTROSOME NUMBER HOMEOSTASIS IN DEVELOPMENT AND DISEASE ......... 1 Introduction .................................................................................................................. 1 Centrioles, Pericentriolar Material, and Centrosome Self-Assembly ........................ 2 Centrosome Number Homeostasis and its Loss during Development and Disease ............................................................................................................................... 14 Centrosome Amplification in Cancer .......................................................................... 20 Centrosome Aberrations in Solid and Hematological Tumors ................................ 21 Aggressive Breast Cancer Cells possess Increased Number of Centrioles and Centrosome Amplification ...................................................................................... 25 Causes of Centrosome Amplification ......................................................................... 27 Cell Cycle Aberrations ............................................................................................ 27 Centriole Overduplication ....................................................................................... 29 Consequences of Centrosome Amplification ............................................................. 30 Genome Instability ................................................................................................. 30 Centrosome Amplification in Tumorigenesis .......................................................... 33 Thesis Outline ............................................................................................................ 38 Materials and Methods for Chapter I.......................................................................... 39 Cell Culture ............................................................................................................ 39 Immunofluorescence .............................................................................................. 39 vi Microscopy ............................................................................................................. 40 Centriole and Centrosome Number Counts ........................................................... 40 Statistics and Biological Replicates ........................................................................ 41 CEP135 ISOFORM DYSREGULATION PROMOTES CENTROSOME AMPLIFICATION IN BREAST CANCER CELLS ....................................................................................... 42 Introduction ................................................................................................................ 42 Results ....................................................................................................................... 48 Centriole Overduplication contributes to Centrosome Amplification in Breast Cancer Cells ....................................................................................................................... 48 The CEP135full:mini Ratio is Elevated in Centrosome Amplified Breast Cancer Cells ............................................................................................................................... 52 Elevated CEP135full is Sufficient to Increase Centrosome Amplification in Breast Cancer Cells........................................................................................................... 57 Elevated CEP135mini Limits the Centriolar Levels of Essential Duplication Factors 60 Elevated CEP135mini is Sufficient to Repress Centrosome Frequency in Breast Cancer Cells........................................................................................................... 62 Mutations Affecting CEP135mini Alternative Polyadenylation Reduce the CEP135full:mini Ratio and Centrosome Amplification ................................................ 64 Discussion ................................................................................................................. 71 Centriole Overduplication in Breast Cancer ........................................................... 71 vii CEP135 Isoform Dysregulation Promotes Centriole Overduplication in Breast Cancer ................................................................................................................... 72 CEP135mini in Repression of Centriole Duplication................................................. 73 Alternative Polyadenylation in CEP135 Isoform Regulation ................................... 74 Materials and Methods for Chapter II ........................................................................
Load more

Recommended publications
  • Genomic Discovery of an Evolutionarily Programmed Modality for Small-Molecule Targeting of an Intractable Protein Surface
    Genomic discovery of an evolutionarily programmed modality for small-molecule targeting of an intractable protein surface Uddhav K. Shigdela,1,2, Seung-Joo Leea,1,3, Mathew E. Sowaa,1,4, Brian R. Bowmana,1,5, Keith Robisona,6, Minyun Zhoua,7, Khian Hong Puaa,8, Dylan T. Stilesa,6, Joshua A. V. Blodgetta,9, Daniel W. Udwarya,10, Andrew T. Rajczewskia,11, Alan S. Manna,12, Siavash Mostafavia,13, Tara Hardyb, Sukrat Aryab,14, Zhigang Wenga,15, Michelle Stewarta,16, Kyle Kenyona,6, Jay P. Morgensterna,6, Ende Pana,17, Daniel C. Graya,6, Roy M. Pollocka,4, Andrew M. Fryb, Richard D. Klausnerc,18, Sharon A. Townsona,19, and Gregory L. Verdinea,d,e,f,2,18,20 Contributed by Richard D. Klausner, April 21, 2020 (sent for review April 8, 2020; reviewed by Chuan He and Ben Shen) The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional Author contributions: U.K.S., S.-J.L., M.E.S., B.R.B., K.R., Z.W., M.S., D.C.G., R.M.P., A.M.F., R.D.K., S.A.T., and G.L.V. designed research; U.K.S., S.-J.L., M.E.S., K.R., M.Z., K.H.P., D.T.S., therapeutic modalities are needed to overcome this deficiency. J.A.V.B., D.W.U., A.T.R., A.S.M., S.M., T.H., S.A., K.K., J.P.M., E.P., R.D.K., and G.L.V. performed Here, the identification and characterization of a natural product, research; M.E.S., D.T.S., and A.M.F.
    [Show full text]
  • Polo-Like Kinase 1 Regulates Nlp, a Centrosome Protein Involved in Microtubule Nucleation
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Developmental Cell, Vol. 5, 113–125, July, 2003, Copyright 2003 by Cell Press Polo-like Kinase 1 Regulates Nlp, a Centrosome Protein Involved in Microtubule Nucleation Martina Casenghi,1,2 Patrick Meraldi,1,2,3 Rieder, 1999; Palazzo et al., 2000). Although centrosome Ulrike Weinhart,1 Peter I. Duncan,1,4 maturation is important for mitotic spindle formation, the Roman Ko¨ rner,1 and Erich A. Nigg1,* underlying mechanisms remain largely unknown. Two 1Department of Cell Biology protein kinases, Polo-like kinase 1 (Plk1; Lane and Nigg, Max Planck Institute of Biochemistry 1996; Sunkel and Glover, 1988) and Aurora-A (Berdnik Am Klopferspitz 18a and Knoblich, 2002; Hannak et al., 2001), as well as D-82152 Martinsried protein phosphatase 4 (Helps et al., 1998; Sumiyoshi Germany et al., 2002), have been implicated in the regulation of centrosome maturation, but the substrates of these en- zymes await identification. Also acting at the G2/M tran- sition, the protein kinase Nek2 and a member of the Summary phosphatase 1 family contribute to regulate centrosome separation, in part through phosphorylation of the centri- In animal cells, most microtubules are nucleated at ole-associated protein C-Nap1 (Fry et al., 1998b; Helps centrosomes. At the onset of mitosis, centrosomes et al., 2000; Mayor et al., 2000). undergo a structural reorganization, termed matura- The discovery of ␥-tubulin and ␥-tubulin-containing tion, which leads to increased microtubule nucleation multiprotein complexes has greatly advanced our un- activity.
    [Show full text]
  • Characterization of NIP2/Centrobin, a Novel Substrate of Nek2, and Its Potential Role in Microtubule Stabilization
    2106 Research Article Characterization of NIP2/centrobin, a novel substrate of Nek2, and its potential role in microtubule stabilization Yeontae Jeong, Jungmin Lee, Kyeongmi Kim, Jae Cheal Yoo and Kunsoo Rhee* Department of Biological Sciences and Research Center for Functional Cellulomics, Seoul National University, Seoul 151-742, Korea *Author for correspondence (e-mail: [email protected]) Accepted 11 April 2007 Journal of Cell Science 120, 2106-2116 Published by The Company of Biologists 2007 doi:10.1242/jcs.03458 Summary Nek2 is a mitotic kinase whose activity varies during the associated with microtubules. Knockdown of NIP2 showed cell cycle. It is well known that Nek2 is involved in significant reduction of microtubule organizing activity, centrosome splitting, and a number of studies have cell shrinkage, defects in spindle assembly and abnormal indicated that Nek2 is crucial for maintaining the integrity nuclear morphology. Based on our results, we propose that of centrosomal structure and microtubule nucleation NIP2 has a role in stabilizing the microtubule structure. activity. In the present study, we report that NIP2, Phosphorylation may be crucial for mobilization of the previously identified as centrobin, is a novel substrate of protein to a new microtubule and stabilizing it. Nek2. NIP2 was daughter-centriole-specific, but was also found in association with a stable microtubule network of cytoplasm. Ectopic NIP2 formed aggregates but was Key words: NIP2, Centrobin, Nek2, Centrosome, Mitosis, dissolved by Nek2 into small pieces and eventually Microtubule Introduction through the phosphorylation-induced displacement of C-Nap1 Phosphorylation is one of major control mechanisms used from the centriolar ends (reviewed in Fry, 2002).
    [Show full text]
  • Spink2 Modulates Apoptotic Susceptibility and Is a Candidate Gene in the Rgcs1 QTL That Affects Retinal Ganglion Cell Death After Optic Nerve Damage
    Spink2 Modulates Apoptotic Susceptibility and Is a Candidate Gene in the Rgcs1 QTL That Affects Retinal Ganglion Cell Death after Optic Nerve Damage Joel A. Dietz1., Margaret E. Maes1., Shuang Huang2, Brian S. Yandell2, Cassandra L. Schlamp1, Angela D. Montgomery1, R. Rand Allingham3, Michael A. Hauser3, Robert W. Nickells1* 1 Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin, United States of America, 2 Department of Biostatistics, University of Wisconsin, Madison, Wisconsin, United States of America, 3 Center for Human Genetics, Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America Abstract The Rgcs1 quantitative trait locus, on mouse chromosome 5, influences susceptibility of retinal ganglion cells to acute damage of the optic nerve. Normally resistant mice (DBA/2J) congenic for the susceptible allele from BALB/cByJ mice exhibit susceptibility to ganglion cells, not only in acute optic nerve crush, but also to chronic inherited glaucoma that is characteristic of the DBA/2J strain as they age. SNP mapping of this QTL has narrowed the region of interest to 1 Mb. In this region, a single gene (Spink2) is the most likely candidate for this effect. Spink2 is expressed in retinal ganglion cells and is increased after optic nerve damage. This gene is also polymorphic between resistant and susceptible strains, containing a single conserved amino acid change (threonine to serine) and a 220 bp deletion in intron 1 that may quantitatively alter endogenous expression levels between strains. Overexpression of the different variants of Spink2 in D407 tissue culture cells also increases their susceptibility to the apoptosis-inducing agent staurosporine in a manner consistent with the differential susceptibility between the DBA/2J and BALB/cByJ strains.
    [Show full text]
  • Cep57 and Cep57l1 Function Redundantly to Recruit the Cep63
    © 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs241836. doi:10.1242/jcs.241836 SHORT REPORT Cep57 and Cep57l1 function redundantly to recruit the Cep63–Cep152 complex for centriole biogenesis Huijie Zhao1,*, Sen Yang1,2,*, Qingxia Chen1,2,3, Xiaomeng Duan1, Guoqing Li1, Qiongping Huang1, Xueliang Zhu1,2,3,‡ and Xiumin Yan1,‡ ABSTRACT SAS-5 in Caenorhabditis elegans) to load Sas-6 for cartwheel The Cep63–Cep152 complex located at the mother centriole recruits formation (Arquint et al., 2015, 2012; Cizmecioglu et al., 2010; Plk4 to initiate centriole biogenesis. How the complex is targeted to Dzhindzhev et al., 2010; Moyer et al., 2015; Ohta et al., 2014, 2018). mother centrioles, however, is unclear. In this study, we show that Several proteins, including Cep135, Cpap (also known as CENPJ), Cep57 and its paralog, Cep57l1, colocalize with Cep63 and Cep152 Cp110 (CCP110) and Cep120, contribute to building the centriolar at the proximal end of mother centrioles in both cycling cells and microtubule (MT) wall and mediate centriole elongation (Azimzadeh multiciliated cells undergoing centriole amplification. Both Cep57 and and Marshall, 2010; Brito et al., 2012; Carvalho-Santos et al., 2012; Cep57l1 bind to the centrosomal targeting region of Cep63. The Comartin et al., 2013; Hung et al., 2004; Kohlmaier et al., 2009; Lin depletion of both proteins, but not either one, blocks loading of the et al., 2013a,b; Loncarek and Bettencourt-Dias, 2018; Schmidt et al., Cep63–Cep152 complex to mother centrioles and consequently 2009). It is known that Cep152 is recruited by Cep63 to act as the cradle prevents centriole duplication.
    [Show full text]
  • A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family
    Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2018 A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family Linda Molla Follow this and additional works at: https://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Linda Molla June 2018 © Copyright by Linda Molla 2018 A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY Linda Molla, Ph.D. The Rockefeller University 2018 APOBEC2 is a member of the AID/APOBEC cytidine deaminase family of proteins. Unlike most of AID/APOBEC, however, APOBEC2’s function remains elusive. Previous research has implicated APOBEC2 in diverse organisms and cellular processes such as muscle biology (in Mus musculus), regeneration (in Danio rerio), and development (in Xenopus laevis). APOBEC2 has also been implicated in cancer. However the enzymatic activity, substrate or physiological target(s) of APOBEC2 are unknown. For this thesis, I have combined Next Generation Sequencing (NGS) techniques with state-of-the-art molecular biology to determine the physiological targets of APOBEC2. Using a cell culture muscle differentiation system, and RNA sequencing (RNA-Seq) by polyA capture, I demonstrated that unlike the AID/APOBEC family member APOBEC1, APOBEC2 is not an RNA editor. Using the same system combined with enhanced Reduced Representation Bisulfite Sequencing (eRRBS) analyses I showed that, unlike the AID/APOBEC family member AID, APOBEC2 does not act as a 5-methyl-C deaminase.
    [Show full text]
  • Novel Targets of Apparently Idiopathic Male Infertility
    International Journal of Molecular Sciences Review Molecular Biology of Spermatogenesis: Novel Targets of Apparently Idiopathic Male Infertility Rossella Cannarella * , Rosita A. Condorelli , Laura M. Mongioì, Sandro La Vignera * and Aldo E. Calogero Department of Clinical and Experimental Medicine, University of Catania, 95123 Catania, Italy; [email protected] (R.A.C.); [email protected] (L.M.M.); [email protected] (A.E.C.) * Correspondence: [email protected] (R.C.); [email protected] (S.L.V.) Received: 8 February 2020; Accepted: 2 March 2020; Published: 3 March 2020 Abstract: Male infertility affects half of infertile couples and, currently, a relevant percentage of cases of male infertility is considered as idiopathic. Although the male contribution to human fertilization has traditionally been restricted to sperm DNA, current evidence suggest that a relevant number of sperm transcripts and proteins are involved in acrosome reactions, sperm-oocyte fusion and, once released into the oocyte, embryo growth and development. The aim of this review is to provide updated and comprehensive insight into the molecular biology of spermatogenesis, including evidence on spermatogenetic failure and underlining the role of the sperm-carried molecular factors involved in oocyte fertilization and embryo growth. This represents the first step in the identification of new possible diagnostic and, possibly, therapeutic markers in the field of apparently idiopathic male infertility. Keywords: spermatogenetic failure; embryo growth; male infertility; spermatogenesis; recurrent pregnancy loss; sperm proteome; DNA fragmentation; sperm transcriptome 1. Introduction Infertility is a widespread condition in industrialized countries, affecting up to 15% of couples of childbearing age [1]. It is defined as the inability to achieve conception after 1–2 years of unprotected sexual intercourse [2].
    [Show full text]
  • The Genetic Program of Pancreatic Beta-Cell Replication in Vivo
    Page 1 of 65 Diabetes The genetic program of pancreatic beta-cell replication in vivo Agnes Klochendler1, Inbal Caspi2, Noa Corem1, Maya Moran3, Oriel Friedlich1, Sharona Elgavish4, Yuval Nevo4, Aharon Helman1, Benjamin Glaser5, Amir Eden3, Shalev Itzkovitz2, Yuval Dor1,* 1Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel 2Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. 3Department of Cell and Developmental Biology, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 4Info-CORE, Bioinformatics Unit of the I-CORE Computation Center, The Hebrew University and Hadassah, The Institute for Medical Research Israel- Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel 5Endocrinology and Metabolism Service, Department of Internal Medicine, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel *Correspondence: [email protected] Running title: The genetic program of pancreatic β-cell replication 1 Diabetes Publish Ahead of Print, published online March 18, 2016 Diabetes Page 2 of 65 Abstract The molecular program underlying infrequent replication of pancreatic beta- cells remains largely inaccessible. Using transgenic mice expressing GFP in cycling cells we sorted live, replicating beta-cells and determined their transcriptome. Replicating beta-cells upregulate hundreds of proliferation- related genes, along with many novel putative cell cycle components. Strikingly, genes involved in beta-cell functions, namely glucose sensing and insulin secretion were repressed. Further studies using single molecule RNA in situ hybridization revealed that in fact, replicating beta-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in beta-cell function.
    [Show full text]
  • C-Nap1 Mutation Affects Centriole Cohesion and Is Associated with a Seckel-Like Syndrome in Cattle
    ARTICLE Received 27 Jul 2014 | Accepted 11 Mar 2015 | Published 23 Apr 2015 DOI: 10.1038/ncomms7894 OPEN C-Nap1 mutation affects centriole cohesion and is associated with a Seckel-like syndrome in cattle Sandrine Floriot1, Christine Vesque2,3,4, Sabrina Rodriguez1,5, Florence Bourgain-Guglielmetti2,3,6, Anthi Karaiskou2,3, Mathieu Gautier1,7, Amandine Duchesne1, Sarah Barbey8,Se´bastien Fritz1,9, Alexandre Vasilescu10, Maud Bertaud1, Mohammed Moudjou11, Sophie Halliez11, Vale´rie Cormier-Daire12, Joyce E.L. Hokayem12, Erich A. Nigg13, Luc Manciaux14,w, Raphae¨l Guatteo15,16, Nora Cesbron15,16, Geraldine Toutirais17, Andre´ Eggen1, Sylvie Schneider-Maunoury2,3,4, Didier Boichard1, Joelle Sobczak-The´pot2,3,6 & Laurent Schibler1,9 Caprine-like Generalized Hypoplasia Syndrome (SHGC) is an autosomal-recessive disorder in Montbe´liarde cattle. Affected animals present a wide range of clinical features that include the following: delayed development with low birth weight, hind limb muscular hypoplasia, caprine-like thin head and partial coat depigmentation. Here we show that SHGC is caused by a truncating mutation in the CEP250 gene that encodes the centrosomal protein C-Nap1. This mutation results in centrosome splitting, which neither affects centriole ultrastructure and duplication in dividing cells nor centriole function in cilium assembly and mitotic spindle organization. Loss of C-Nap1-mediated centriole cohesion leads to an altered cell migration phenotype. This discovery extends the range of loci that constitute the spectrum of autosomal primary recessive microcephaly (MCPH) and Seckel-like syndromes. 1 Institut National de la Recherche Agronomique (INRA), Unite´ Mixte de Recherche 1313—Ge´ne´tique Animale et Biologie Inte´grative (UMR1313—GABI), F-78352 Jouy-en-Josas, France.
    [Show full text]
  • Multicilin Drives Centriole Biogenesis Via E2f Proteins
    Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Multicilin drives centriole biogenesis via E2f proteins Lina Ma,1 Ian Quigley,1 Heymut Omran,2 and Chris Kintner1,3 1The Salk Institute for Biological Studies, La Jolla 92037, California, USA; 2Department of Pediatrics, University Hospital Muenster, 48149 Muenster; Germany Multiciliate cells employ hundreds of motile cilia to produce fluid flow, which they nucleate and extend by first assembling hundreds of centrioles. In most cells, entry into the cell cycle allows centrioles to undergo a single round of duplication, but in differentiating multiciliate cells, massive centriole assembly occurs in G0 by a process initiated by a small coiled-coil protein, Multicilin. Here we show that Multicilin acts by forming a ternary complex with E2f4 or E2f5 and Dp1 that binds and activates most of the genes required for centriole biogenesis, while other cell cycle genes remain off. This complex also promotes the deuterosome pathway of centriole biogenesis by activating the expression of deup1 but not its paralog, cep63. Finally, we show that this complex is disabled by mutations in human Multicilin that cause a severe congenital mucociliary clearance disorder due to reduced generation of multiple cilia. By coopting the E2f regulation of cell cycle genes, Multicilin drives massive centriole assembly in epithelial progenitors in a manner required for multiciliate cell differentiation. [Keywords: centrioles; e2f4; multiciliate cells] Supplemental material is available for this article. Received April 17, 2014; revised version accepted May 27, 2014. The centriole, a cylindrical organelle made up of triplet cently have come into focus molecularly (Tang 2013).
    [Show full text]
  • Plk4-Induced Centriole Biogenesis in Human Cells
    Plk4-induced Centriole Biogenesis in Human Cells Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften der Fakultät für Biologie der Ludwig-Maximilians Universität München Vorgelegt von Julia Kleylein-Sohn München, 2007 Dissertation eingereicht am: 27.11.2007 Tag der mündlichen Prüfung: 18.04.2008 Erstgutachter: Prof. E. A. Nigg Zweitgutachter: PD Dr. Angelika Böttger 2 Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig und ohne unerlaubte Hilfe angefertigt habe. Sämtliche Experimente wurden von mir selbst durchgeführt, soweit nicht explizit auf Dritte verwiesen wird. Ich habe weder an anderer Stelle versucht, eine Dissertation oder Teile einer solchen einzureichen bzw. einer Prüfungskommission vorzulegen, noch eine Doktorprüfung zu absolvieren. München, den 22.11.2007 3 TABLE OF CONTENTS SUMMARY…………………………………………………………………………..………. 6 INTRODUCTION……………………………………………………………………………. 7 Structure of the centrosome…………………………………………………………….. 7 The centrosome cycle…………………………………………………………………..10 Kinases involved in the regulation of centriole duplication………………………….12 Maintenance of centrosome numbers………………………………………………...13 Licensing of centriole duplication……………………………………………………... 15 ‘De novo ’ centriole assembly pathways in mammalian cells…………………..…...15 Templated centriole biogenesis in mammalian cells……………………………….. 18 The role of centrins and Sfi1p in centrosome duplication ……………………...…..19 Centriole biogenesis in C. elegans …………………………………………………… 21 Centriole biogenesis in human cells………………………………………………….. 23 Centrosome
    [Show full text]
  • A SARS-Cov-2 Protein Interaction Map Reveals Targets for Drug Repurposing
    Article A SARS-CoV-2 protein interaction map reveals targets for drug repurposing https://doi.org/10.1038/s41586-020-2286-9 A list of authors and affiliations appears at the end of the paper Received: 23 March 2020 Accepted: 22 April 2020 A newly described coronavirus named severe acute respiratory syndrome Published online: 30 April 2020 coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than Check for updates 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efcacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and eforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identifed the human proteins that physically associated with each of the SARS-CoV-2 proteins using afnity-purifcation mass spectrometry, identifying 332 high-confdence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors.
    [Show full text]