DOCSLIB.ORG

HSET Complex Links Centrosomes with Spindle Poles and Drives Centrosome Clustering in Cancer

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
HSET Complex Links Centrosomes with Spindle Poles and Drives Centrosome Clustering in Cancer ARTICLE Received 10 Sep 2015 | Accepted 10 Feb 2016 | Published 18 Mar 2016 DOI: 10.1038/ncomms11005 OPEN A CEP215–HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer Pavithra L. Chavali1, Gayathri Chandrasekaran1, Alexis R. Barr1,w,Pe´ter Ta´trai1, Chris Taylor1, Evaggelia K. Papachristou1, C. Geoffrey Woods2, Sreenivas Chavali3 & Fanni Gergely1 Numerical centrosome aberrations underlie certain developmental abnormalities and may promote cancer. A cell maintains normal centrosome numbers by coupling centrosome duplication with segregation, which is achieved through sustained association of each centrosome with a mitotic spindle pole. Although the microcephaly- and primordial dwarfism- linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic profiling, we identify the minus end-directed microtubule motor protein HSET as a direct binding partner of CEP215. Targeted deletion of the HSET-binding domain of CEP215 in vertebrate cells causes centro- some detachment and results in HSET depletion at centrosomes, a phenotype also observed in CEP215-deficient patient-derived cells. Moreover, in cancer cells with centrosome amplification, the CEP215–HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosome–spindle pole interface by the CEP215–HSET complex could promote survival of cancer cells containing supernumerary centrosomes. 1 Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK. 2 Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK. 3 MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. w Present address: Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK. Correspondence and requests for materials should be addressed to F.G. (email: [email protected]). NATURE COMMUNICATIONS | 7:11005 | DOI: 10.1038/ncomms11005 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11005 entrosomes act as dominant sites of microtubule assembly To this end, affinity purification tags (GsTAP containing protein in mitosis and therefore centrosome number corresponds G and streptavidin-binding protein) were inserted in-frame into Cto the number of spindle poles formed1. Because faithful both alleles of the CEP215 gene (CEP215-TAP cell line) transmission of genetic information requires a bipolar mitotic in the chicken B cell line, DT40 (refs 27,35). Following spindle, centrosome numbers must be tightly controlled in affinity purification, protein complexes were analysed by mass cells. Accordingly, centrosome numbers are regulated by two spectrometry (Fig. 1a; Supplementary Fig. 1). Proteins were mechanisms. First, centrosome duplication is limited to once per considered as hits if they were represented by one or more unique cell cycle ensuring that cells enter mitosis with two functional peptides in all three biological replicates and by four or more centrosomes2,3. Second, each centrosome associates and unique peptides in at least two replicates. We filtered out putative co-segregates with its own mitotic spindle pole causing each hits if they were represented even by a single unique peptide in daughter cell to inherit precisely one centrosome4. Centrosomes pulldowns performed from wild-type (WT) cells. Hits were and mitotic spindle poles are distinct structures, well illustrated further filtered against other GsTAP affinity purification experi- by the presence of focused spindle poles in cells lacking ments to exclude TAP tag-specific binding36. An interacting centrosomes5–7. Spindle pole formation relies on microtubule network of CEP215 was constructed based on these criteria motors and microtubule-associated proteins that crosslink and (Fig. 1b). All previously reported interacting partners have focus bundles of kinetochore-associated microtubules (k-fibres). been identified, in addition to new ones that include PCM1, In Drosophila S2 cells the key protein responsible for holding CKAP5/ch-Tog and HSET, a minus end-directed microtubule centrosomes at spindle poles is dynein, a minus end-directed motor (Fig. 1b; Supplementary Table 1; Supplementary Data 1). motor8–10. Dynactin increases the processivity of dynein and Western blot analysis confirmed interactions (Fig. 1c). Because of together they transport the spindle pole integrity protein, nuclear its roles in mitotic spindle pole organization in Drosophila and mitotic apparatus (NuMA) to the minus ends of spindle cancer cells, we have decided to focus on HSET for the purpose of microtubules11,12. In NuMA-deficient mammalian cells, k-fibres this study. lose focus and centrosomes detach from the poles13. Similar phenotypes have been documented in Drosophila cells and CEP215 and HSET bind directly in vertebrates. CEP215 embryos upon disruption of the minus end-directed kinesin-14 interacts with the microtubule motor dynein and its adaptor, motor protein, non-claret-disjunctional (ncd)10,14. By contrast, dynactin37. To establish if HSET, dynein and CEP215 exist in the the mammalian homologue HSET is largely dispensable for same complex, CEP215-TAP-containing protein complexes k-fibre focus. Instead, HSET contributes to spindle elongation were fractionated on a sucrose gradient. CEP215-bound HSET through crosslinking and sliding microtubules, functions sedimented at a lower sucrose concentration than CEP215-bound dependent on its C-terminal motor domain and the additional dynein, indicative of separate complexes (Fig. 2a). Gel filtration microtubule-binding site in its N-terminal tail15. Both ncd experiment yielded similar results (Supplementary Fig. 2a). and HSET have been implicated in survival of cells with To further characterize the CEP215-HSET interaction, we centrosome amplification16–19. In particular, the orthologues elucidated the respective binding domains in human CEP215 and mediate clustering of supernumerary centrosomes into pseudo- HSET. CEP215 fusion products were expressed in HeLa cells bipolar spindles, a role essential for continued proliferation of constitutively depleted of CEP215 (Supplementary Fig. 2b). The cells with centrosome amplification. HSET also promotes HSET-binding region was mapped to the two overlapping regions clustering of acentrosomal spindle poles17. in the N terminus of CEP215: amino acids (aa) 500–700 and 300– The centrosome comprises a pair of centrioles embedded 600 (Fig. 2b,c). In HSET it is aa1–150 at the N terminus (that is, in the pericentriolar matrix (PCM), the site of microtubule the tail domain) that binds CEP215 (Fig. 2d). The CEP215–HSET nucleation. CEP215 is an evolutionarily conserved PCM interaction is direct, as suggested by yeast two-hybrid assays and protein present in microtubule-organizing centres from yeast to surface plasmon resonance (SPR) (Fig. 2e; Supplementary human; the centrosomin motif 1 (CM1) in its N terminus binds Fig. 2c). In SPR aa500–700 of CEP215 displayed an B2.5-fold the g-tubulin complex20–23. CEP215 organizes several PCM greater binding to HSET when compared with aa300–600. components including pericentrin and AKAP450 (refs 24–30). We therefore consider aa500–700 of CEP215 as the minimal Deletion of centrosomin (cnn), its Drosophila orthologue, HSET-binding region (HBR). Sequence analysis of HBR of disruption of the CM1 domain of chicken CEP215 and human CEP215 revealed three helical regions that are conserved depletion of CEP215 in HeLa cells all cause centrosome in vertebrates. Remarkably, the tail of HSET also shows a high detachment from mitotic spindle poles27,31,32. However, spindle degree of conservation in the vertebrate lineage, raising the pole focus is maintained in CM1-deficient cells, consistent with possibility that the interaction between HSET and CEP215 arose normal localization of NuMA and dynactin27. Mutations in in this lineage (Fig. 2f; Supplementary Figs 2d, 3 and 4). Indeed, CEP215 are associated with congenital diseases such as primary we could not detect binding between Drosophila cnn and ncd, the microcephaly and primordial dwarfism33,34. respective homologues of human CEP215 and HSET, whereas Here we set out to identify the molecular mechanism by which the two proteins co-immunoprecipitated in human HeLa cells CEP215 maintains centrosome attachment to spindle poles. We (Fig. 2g,h). The ancestral cnn gene underwent a duplication identify HSET as a direct interactor of CEP215 and demonstrate event in cephalochordates producing CEP215 and another that HSET binding by CEP215 is crucial for its role in this CM1-containing gene, myomegalin. Unlike CEP215, myomegalin process. We further show that cancer cells with centrosome lacks an HBR and, accordingly, failed to interact with HSET amplification rely on the CEP215–HSET complex for centrosome (Supplementary Fig. 2e). clustering and survival. CEP215-HSET complex connects centrosomes to spindle poles. Results We next wanted to address the functional significance of the Identification of CEP215-interacting partners in DT40 cells. CEP215–HSET interaction. Using gene targeting we created To establish the molecular basis for CEP215 function in chicken DT40 cell lines in which either HSET or the HBR of centrosome–spindle pole attachment, we employed an unbiased CEP215 was disrupted.
Load more

Recommended publications
  • Title a New Centrosomal Protein Regulates Neurogenesis By
    Title A new centrosomal protein regulates neurogenesis by microtubule organization Authors: Germán Camargo Ortega1-3†, Sven Falk1,2†, Pia A. Johansson1,2†, Elise Peyre4, Sanjeeb Kumar Sahu5, Loïc Broic4, Camino De Juan Romero6, Kalina Draganova1,2, Stanislav Vinopal7, Kaviya Chinnappa1‡, Anna Gavranovic1, Tugay Karakaya1, Juliane Merl-Pham8, Arie Geerlof9, Regina Feederle10,11, Wei Shao12,13, Song-Hai Shi12,13, Stefanie M. Hauck8, Frank Bradke7, Victor Borrell6, Vijay K. Tiwari§, Wieland B. Huttner14, Michaela Wilsch- Bräuninger14, Laurent Nguyen4 and Magdalena Götz1,2,11* Affiliations: 1. Institute of Stem Cell Research, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany. 2. Physiological Genomics, Biomedical Center, Ludwig-Maximilian University Munich, Germany. 3. Graduate School of Systemic Neurosciences, Biocenter, Ludwig-Maximilian University Munich, Germany. 4. GIGA-Neurosciences, Molecular regulation of neurogenesis, University of Liège, Belgium 5. Institute of Molecular Biology (IMB), Mainz, Germany. 6. Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas and Universidad Miguel Hernández, Sant Joan d’Alacant, Spain. 7. Laboratory for Axon Growth and Regeneration, German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany. 8. Research Unit Protein Science, Helmholtz Centre Munich, German Research Center for Environmental Health, Munich, Germany. 9. Protein Expression and Purification Facility, Institute of Structural Biology, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany. 10. Institute for Diabetes and Obesity, Monoclonal Antibody Core Facility, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany. 11. SYNERGY, Excellence Cluster of Systems Neurology, Biomedical Center, Ludwig- Maximilian University Munich, Germany. 12. Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, USA 13.
    [Show full text]
  • Mutations in CDK5RAP2 Cause Seckel Syndrome Go¨ Khan Yigit1,2,3,A, Karen E
    ORIGINAL ARTICLE Mutations in CDK5RAP2 cause Seckel syndrome Go¨ khan Yigit1,2,3,a, Karen E. Brown4,a,Hu¨ lya Kayserili5, Esther Pohl1,2,3, Almuth Caliebe6, Diana Zahnleiter7, Elisabeth Rosser8, Nina Bo¨ gershausen1,2,3, Zehra Oya Uyguner5, Umut Altunoglu5, Gudrun Nu¨ rnberg2,3,9, Peter Nu¨ rnberg2,3,9, Anita Rauch10, Yun Li1,2,3, Christian Thomas Thiel7 & Bernd Wollnik1,2,3 1Institute of Human Genetics, University of Cologne, Cologne, Germany 2Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany 3Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany 4Chromosome Biology Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, W12 0NN, UK 5Department of Medical Genetics, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey 6Institute of Human Genetics, Christian-Albrechts-University of Kiel, Kiel, Germany 7Institute of Human Genetics, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany 8Department of Clinical Genetics, Great Ormond Street Hospital for Children, London, WC1N 3EH, UK 9Cologne Center for Genomics, University of Cologne, Cologne, Germany 10Institute of Medical Genetics, University of Zurich, Schwerzenbach-Zurich, Switzerland Keywords Abstract CDK5RAP2, CEP215, microcephaly, primordial dwarfism, Seckel syndrome Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by pre- natal proportionate short stature, severe microcephaly, intellectual disability, and Correspondence characteristic facial features. Here, we describe the novel homozygous splice-site Bernd Wollnik, Center for Molecular mutations c.383+1G>C and c.4005-9A>GinCDK5RAP2 in two consanguineous Medicine Cologne (CMMC) and Institute of families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal pro- Human Genetics, University of Cologne, tein which is known to be essential for centrosomal cohesion and proper spindle Kerpener Str.
    [Show full text]
  • The Ciliated Cell Transcriptome A
    THE CILIATED CELL TRANSCRIPTOME A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL SCIENCES AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Ramona Hoh March 2010 ! © 2010 by Ramona Amy Hoh. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/sk794dv5857 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Timothy Stearns, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Mark Krasnow I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Maxence Nachury I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. William Nelson Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives.
    [Show full text]
  • Bioinformatics Identi Cation of Prognostic Factors Associated With
    Bioinformatics Identication of Prognostic Factors Associated with Breast Cancer Ying Wei Sichuan University https://orcid.org/0000-0001-8178-4705 Shipeng Zhang College of Pharmacy, North Sichuan Medical College Li Xiao West China School of Basic Medical Sciences and Forensic Medicine Jing Zou West China School of Basic Medical Sciences and Forensic Medicine Yingqing Fu West China School of Basic Medical Sciences and Forensic Medicine Yi Ye West China School of Basic Medical Sciences and Forensic Medicine Linchuan Liao ( [email protected] ) West China School of Basic Medical Sciences and Forensic Medicine https://orcid.org/0000-0003-3700-8471 Research Keywords: Breast cancer, Differentially expressed genes, miRNAs, Transcription factors, Bioinformatic analysis Posted Date: December 2nd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-117477/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/23 Abstract Background: Breast cancer (BRCA) remains one of the most common forms of cancer and is the most prominent driver of cancer-related death among women. The mechanistic basis for BRCA, however, remains incompletely understood. In particular, the relationships between driver mutations and signaling pathways in BRCA are poorly characterized, making it dicult to identify reliable clinical biomarkers that can be employed in diagnostic, therapeutic, or prognostic contexts. Methods: First, we downloaded publically available BRCA datasets (GSE45827, GSE42568, and GSE61304) from the Gene Expression Omnibus (GEO) database. We then compared gene expression proles between tumor and control tissues in these datasets using Venn diagrams and the GEO2R analytical tool. We further explore the functional relevance of BRCA-associated differentially expressed genes (DEGs) via functional and pathway enrichment analyses using the DAVID tool, and we then constructed a protein-protein interaction network incorporating DEGs of interest using the Search Tool for the Retrieval of Interacting Genes (STRING) database.
    [Show full text]
  • Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-Like Mouse Models: Tracking the Role of the Hairless Gene
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2006 Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-like Mouse Models: Tracking the Role of the Hairless Gene Yutao Liu University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Life Sciences Commons Recommended Citation Liu, Yutao, "Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino- like Mouse Models: Tracking the Role of the Hairless Gene. " PhD diss., University of Tennessee, 2006. https://trace.tennessee.edu/utk_graddiss/1824 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Yutao Liu entitled "Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-like Mouse Models: Tracking the Role of the Hairless Gene." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Brynn H. Voy, Major Professor We have read this dissertation and recommend its acceptance: Naima Moustaid-Moussa, Yisong Wang, Rogert Hettich Accepted for the Council: Carolyn R.
    [Show full text]
  • The P53/P73 - P21cip1 Tumor Suppressor Axis Guards Against Chromosomal Instability by Restraining CDK1 in Human Cancer Cells
    Oncogene (2021) 40:436–451 https://doi.org/10.1038/s41388-020-01524-4 ARTICLE The p53/p73 - p21CIP1 tumor suppressor axis guards against chromosomal instability by restraining CDK1 in human cancer cells 1 1 2 1 2 Ann-Kathrin Schmidt ● Karoline Pudelko ● Jan-Eric Boekenkamp ● Katharina Berger ● Maik Kschischo ● Holger Bastians 1 Received: 2 July 2020 / Revised: 2 October 2020 / Accepted: 13 October 2020 / Published online: 9 November 2020 © The Author(s) 2020. This article is published with open access Abstract Whole chromosome instability (W-CIN) is a hallmark of human cancer and contributes to the evolvement of aneuploidy. W-CIN can be induced by abnormally increased microtubule plus end assembly rates during mitosis leading to the generation of lagging chromosomes during anaphase as a major form of mitotic errors in human cancer cells. Here, we show that loss of the tumor suppressor genes TP53 and TP73 can trigger increased mitotic microtubule assembly rates, lagging chromosomes, and W-CIN. CDKN1A, encoding for the CDK inhibitor p21CIP1, represents a critical target gene of p53/p73. Loss of p21CIP1 unleashes CDK1 activity which causes W-CIN in otherwise chromosomally stable cancer cells. fi Vice versa 1234567890();,: 1234567890();,: Consequently, induction of CDK1 is suf cient to induce abnormal microtubule assembly rates and W-CIN. , partial inhibition of CDK1 activity in chromosomally unstable cancer cells corrects abnormal microtubule behavior and suppresses W-CIN. Thus, our study shows that the p53/p73 - p21CIP1 tumor suppressor axis, whose loss is associated with W-CIN in human cancer, safeguards against chromosome missegregation and aneuploidy by preventing abnormally increased CDK1 activity.
    [Show full text]
  • Cep57, a NEDD1-Binding Pericentriolar Material Component, Is Essential for Spindle Pole Integrity
    Cell Research (2012) :1-12. © 2012 IBCB, SIBS, CAS All rights reserved 1001-0602/12 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr Cep57, a NEDD1-binding pericentriolar material component, is essential for spindle pole integrity Qixi Wu1, *, Runsheng He1, *, Haining Zhou1, Albert CH Yu2, Bo Zhang1, Junlin Teng1, Jianguo Chen1, 3 1The State Key Laboratory of Biomembrane and Membrane Bioengineering and The Key Laboratory of Cell Proliferation and Dif- ferentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, China; 2Department of Neurobi- ology, Neuroscience Research Institute, School of Basic Medical Sciences, Peking University, Beijing 100191, China; 3The Center for Theoretical Biology, Peking University, Beijing 100871, China Formation of a bipolar spindle is indispensable for faithful chromosome segregation and cell division. Spindle in- tegrity is largely dependent on the centrosome and the microtubule network. Centrosome protein Cep57 can bundle microtubules in mammalian cells. Its related protein (Cep57R) in Xenopus was characterized as a stabilization factor for microtubule-kinetochore attachment. Here we show that Cep57 is a pericentriolar material (PCM) component. Its interaction with NEDD1 is necessary for the centrosome localization of Cep57. Depletion of Cep57 leads to unaligned chromosomes and a multipolar spindle, which is induced by PCM fragmentation. In the absence of Cep57, cen- trosome microtubule array assembly activity is weakened, and the spindle length and microtubule density decrease. As a spindle microtubule-binding protein, Cep57 is also responsible for the proper organization of the spindle micro- tubule and localization of spindle pole focusing proteins. Collectively, these results suggest that Cep57, as a NEDD1- binding centrosome component, could function as a spindle pole- and microtubule-stabilizing factor for establishing robust spindle architecture.
    [Show full text]
  • Anatomy of the Axon
    Anatomy of the Axon: Dissecting the role of microtubule plus-end tracking proteins in axons of hippocampal neurons ISBN: 978-90-393-7095-7 On the cover: Interpretation of Rembrandt van Rijn’s iconic oil painting The Anatomy Lesson of Dr. Nicolaes Tulp (1632). Instead of a human subject, a neuron is being dissected under the watchful eye of members of the Hoogenraad & Akhmanova labs circa 2018. The doctoral candidate takes on the role of Dr. Nicolaes Tulp. The year in which this thesis is published, 2019, marks the 350th anniversary of Rembrandt’s passing. It is therefore declared Year of Rembrandt. Cover photography: Jasper Landman (Bob & Fzbl Photography) Design and layout: Dieudonnée van de Willige © 2019; CC-BY-NC-ND. Printed in The Netherlands. Anatomy of the Axon: Dissecting the role of microtubule plus-end tracking proteins in axons of hippocampal neurons Anatomie van de Axon: Ontleding van de rol van microtubulus-plus-eind-bindende eiwitten in axonen van hippocampale neuronen (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. H.R.B.M. Kummeling, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op woensdag 22 mei 2019 des middags te 2.30 uur door Dieudonnée van de Willige geboren op 24 oktober 1990 te Antwerpen, België Promotoren: Prof. dr. C.C. Hoogenraad Prof. dr. A. Akhmanova Voor Hans & Miranda Index Preface 7 List of non-standard abbreviations 9 Microtubule plus-end tracking proteins
    [Show full text]
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [Show full text]
  • Supplementary Data
    SUPPLEMENTARY DATA A cyclin D1-dependent transcriptional program predicts clinical outcome in mantle cell lymphoma Santiago Demajo et al. 1 SUPPLEMENTARY DATA INDEX Supplementary Methods p. 3 Supplementary References p. 8 Supplementary Tables (S1 to S5) p. 9 Supplementary Figures (S1 to S15) p. 17 2 SUPPLEMENTARY METHODS Western blot, immunoprecipitation, and qRT-PCR Western blot (WB) analysis was performed as previously described (1), using cyclin D1 (Santa Cruz Biotechnology, sc-753, RRID:AB_2070433) and tubulin (Sigma-Aldrich, T5168, RRID:AB_477579) antibodies. Co-immunoprecipitation assays were performed as described before (2), using cyclin D1 antibody (Santa Cruz Biotechnology, sc-8396, RRID:AB_627344) or control IgG (Santa Cruz Biotechnology, sc-2025, RRID:AB_737182) followed by protein G- magnetic beads (Invitrogen) incubation and elution with Glycine 100mM pH=2.5. Co-IP experiments were performed within five weeks after cell thawing. Cyclin D1 (Santa Cruz Biotechnology, sc-753), E2F4 (Bethyl, A302-134A, RRID:AB_1720353), FOXM1 (Santa Cruz Biotechnology, sc-502, RRID:AB_631523), and CBP (Santa Cruz Biotechnology, sc-7300, RRID:AB_626817) antibodies were used for WB detection. In figure 1A and supplementary figure S2A, the same blot was probed with cyclin D1 and tubulin antibodies by cutting the membrane. In figure 2H, cyclin D1 and CBP blots correspond to the same membrane while E2F4 and FOXM1 blots correspond to an independent membrane. Image acquisition was performed with ImageQuant LAS 4000 mini (GE Healthcare). Image processing and quantification were performed with Multi Gauge software (Fujifilm). For qRT-PCR analysis, cDNA was generated from 1 µg RNA with qScript cDNA Synthesis kit (Quantabio). qRT–PCR reaction was performed using SYBR green (Roche).
    [Show full text]
  • AURKA, DLGAP5, TPX2, KIF11 and CKAP5: Five Specific Mitosis-Associated Genes Correlate with Poor Prognosis for Non-Small Cell Lung Cancer Patients
    INTERNATIONAL JOURNAL OF ONCOLOGY 50: 365-372, 2017 AURKA, DLGAP5, TPX2, KIF11 and CKAP5: Five specific mitosis-associated genes correlate with poor prognosis for non-small cell lung cancer patients MARC A. SCHNEIDER1,8, PETROS CHristopoulos2,8, THOMAS MULEY1,8, ARNE WartH5,8, URSULA KLINGMUELLER6,8,9, MICHAEL THOMAS2,8, FELIX J.F. HertH3,8, HENDRIK DIENEMANN4,8, NIKOLA S. MUELLER7,9, FABIAN THEIS7,9 and MICHAEL MEISTER1,8,9 1Translational Research Unit, 2Department of Thoracic Oncology, 3Department of Pneumology and Critical Care Medicine, and 4Department of Surgery, Thoraxklinik at University Hospital Heidelberg, Heidelberg; 5Institute of Pathology, University of Heidelberg, Heidelberg; 6Systems Biology of Signal Transduction, German Cancer Research Center, Heidelberg; 7Cellular Dynamics and Cell Patterning, Max Planck Institute of Biochemistry, Martinsried; 8Translational Lung Research Center Heidelberg (TLRC-H), Member of the German Center for Lung Research (DZL) Heidelberg; 9CancerSys network ‘LungSysII’, Heidelberg, Germany Received October 26, 2016; Accepted December 5, 2016 DOI: 10.3892/ijo.2017.3834 Abstract. The growth of a tumor depends to a certain extent genes AURKA, DLGAP5, TPX2, KIF11 and CKAP5 is asso- on an increase in mitotic events. Key steps during mitosis are ciated with the prognosis of NSCLC patients. the regulated assembly of the spindle apparatus and the sepa- ration of the sister chromatids. The microtubule-associated Introduction protein Aurora kinase A phosphorylates DLGAP5 in order to correctly segregate the chromatids. Its activity and recruitment Lung cancer is globally the leading cause of cancer-related to the spindle apparatus is regulated by TPX2. KIF11 and deaths (1). Non-small cell lung cancer (NSCLC), which accounts CKAP5 control the correct arrangement of the microtubules for more than 80% of all cases, is divided in adenocarcinoma and prevent their degradation.
    [Show full text]
  • Assembly of Centrosomal Proteins and Microtubule Organization Depends on PCM-1
    JCBArticle Assembly of centrosomal proteins and microtubule organization depends on PCM-1 Alexander Dammermann and Andreas Merdes Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, UK he protein PCM-1 localizes to cytoplasmic granules treatment of cells with the microtubule inhibitor nocodazole. known as “centriolar satellites” that are partly enriched Inhibition or depletion of PCM-1 function further disrupted T around the centrosome. We inhibited PCM-1 function the radial organization of microtubules without affecting using a variety of approaches: microinjection of antibodies microtubule nucleation. Loss of microtubule organization into cultured cells, overexpression of a PCM-1 deletion was also observed after centrin or ninein depletion. Our mutant, and specific depletion of PCM-1 by siRNA. All data suggest that PCM-1–containing centriolar satellites are Downloaded from approaches led to reduced targeting of centrin, pericentrin, involved in the microtubule- and dynactin-dependent recruit- and ninein to the centrosome. Similar effects were seen ment of proteins to the centrosome, of which centrin and upon inhibition of dynactin by dynamitin, and after prolonged ninein are required for interphase microtubule organization. jcb.rupress.org Introduction Microtubule organization is essential for directional intra- the centrosomal surface or subsequently released and anchored cellular transport, for the modulation of cell morphology in other places of the cell (Mogensen, 1999). The initial step and locomotion, and for the formation of the spindle apparatus of microtubule nucleation is dependent on the function of during cell division. With the exception of plants, most cells 25S ring complexes of the protein ␥-tubulin and associated on December 31, 2017 organize their microtubule network using specialized structures, proteins (Zheng et al., 1995).
    [Show full text]