DOCSLIB.ORG

Deamplification of Supernumerary

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Deamplification of Supernumerary DEAMPLIFICATION OF SUPERNUMERARY CENTROSOMES BY CENTROSOMAL CLUSTERING By Ezekiel Thomas A Thesis Submitted to the Faculty of The Wilkes Honors College In Partial Fulfillment of the Requirements for the Degree of Bachelor of Arts in Liberal Arts and Sciences With a Concentration in Biology Wilkes Honors College of Florida Atlantic University Jupiter, Florida May 2012 Deamplification of Supernumerary Centrosomes by Centrosomal Clustering By Ezekiel Thomas This thesis was prepared under the direction of the candidate’s thesis advisor, Dr. Nicholas Quintyne, and has been approved by the members of the supervisory committee. It was submitted to the faculty of The Honors College and was accepted in partial fulfillment of the requirements for the Degree of Bachelor of Arts in Liberal Arts and Sciences. SUPERVISORY COMMITTEE: ________________________ ________________________ Dr. Nicholas Quintyne Date ________________________ ________________________ Dr. Michelle Ivey Date ________________________ ________________________ Dr. Jeffrey Buller Date Dean, Wilkes Honors College ii Acknowledgements I would like to thank Dr. Quintyne for his mentoring, guidance, and patience; April Schimmel for maintaining the lab and purchasing numerous antibodies; and Dr. Ivey for her assistance as my second reader. I would also like to thank my family and friends for their support. iii Abstract Author: Ezekiel Thomas Title: Deamplification of Supernumerary Centrosomes by Centrosomal Clustering Institution: Wilkes Honors College of Florida Atlantic University Thesis Advisor: Dr. Nicholas Quintyne Degree: Bachelor of Arts in Liberal Arts and Sciences Concentration: Biology Year: 2012 Supernumerary centrosomes can arise in a cell through a variety of methods. The presence of supernumerary centrosomes has been observed in nearly all types of cancer and promotes chromosomal instability, with rates of incident increasing as the cancer progresses. An oral squamous cell carcinoma line was treated with hydroxyurea to induce supernumerary centrosomes in the cells. NuMA was then knocked down using shRNA to promote centrosomal clustering and bipolar mitotic division in cells with supernumerary centrosomes. Immunofluorescence with an antibody against SAS 6 accurately stained the centrioles for observation. The cells exhibiting supernumerary centrosomes undergoing bipolar mitotic division were studied to look for a possible pattern in centrosomal clustering where the majority of centrosomes are at one pole with a single centrosome at the other pole. Initial results suggest the presence of such a mechanism, which would describe a previously unknown mechanism for cells to deamplify supernumerary centrosomes by centrosomal clustering. iv Table of Contents List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii Introduction ......................................................................................................................... 1 The Centrosome: Function, Structure, and Lifecycle ............................................. 1 Development of Supernumerary Centrosomes ....................................................... 2 Supernumerary Centrosomes and Multipolarity ..................................................... 3 Centrosomal Deamplification ................................................................................. 4 Mechanisms for Centrosomal Clustering ................................................................6 Materials and Methods ........................................................................................................ 8 Cell Culture ............................................................................................................. 8 Immunofluorescence ............................................................................................... 8 Antibodies ............................................................................................................... 9 Transfection .......................................................................................................... 10 Drug Treatments ................................................................................................... 11 Microscopy ........................................................................................................... 11 Results ............................................................................................................................... 12 Induction of Supernumerary Centrosomes ........................................................... 12 Immunofluorescent Methods for Centriole Observation ...................................... 14 Induction of Centrosomal Clustering .................................................................... 17 Discussion ......................................................................................................................... 18 Colcemid and Hydroxyurea Treatments ............................................................... 19 Immunofluorescent Staining of Centrioles During Mitosis .................................. 20 A Mechanism Promoting Preferential Clustering ................................................. 21 Continuing Research ............................................................................................. 22 Conclusion ........................................................................................................................ 22 References ......................................................................................................................... 24 v List of Tables Table 1. Summary of 1° antibodies and successful methods for staining centrioles ........ 14 vi List of Figures Figure 1. Methods of dealing with supernumerary centrosomes ........................................ 6 Figure 2. Treatment to induce supernumerary centrosomes ............................................. 13 Figure 3. Microtubule differences in hydroxyurea and colcemid treated cells ................. 14 Figure 4. Staining of cells using SAS 6 antibody ............................................................. 16 Figure 5. Staining of cells using CEP 250 antibody ......................................................... 17 Figure 6. Preferential centrosomal clustering ................................................................... 18 vii I. Introduction The Centrosome: Function, Structure, and Lifecycle The centrosome is the cellular organelle that acts as the microtubule organizing center (MTOC) in most types of cells (Doxsey, 2001). Through the regulation of microtubules (MTs), the centrosome controls cell shape, cell motility, intracellular transport, and the positioning of organelles. The centrosome also plays a key role during division and is responsible for the formation of the spindle poles, which are vital to proper chromosomal segregation and cleavage plane localization (Nigg, 2002). The structure of the centrosome is divided into two main components, the centrioles and the pericentriolar material (PCM). The centrioles are two barrel-shaped objects aligned at right angles to each other and function to anchor the MTs and recruit the PCM. There is an older centriole having more associated proteins (the "mother centriole") and a newer centriole that doesn't have as many associated proteins (the "daughter centriole"). The PCM contains a variety of proteins, most notably the γ-tubulin ring complex, which acts as a template for new MTs and serves as a site of MT nucleation (Doxsey, 2001). The centrosome cycle is closely related to the chromosomal duplication cycle, and can be divided into five main steps: centriole disorientation, centriole duplication, centriole elongation, centrosome maturation, and centrosome separation (Nigg, 2002). The process begins in late G1 of the cell cycle once the cell has committed to division, and starts with the loss of orientation between the two centrioles. As the cell progresses into S phase where DNA synthesis occurs, the centrioles undergo duplication as well in a semi-conservative fashion from the perspective of the centrosome, while additional 1 pericentriolar material is also recruited (Balczon et al., 1999). This process continues into G2 with centriole elongation. In G2, as elongation continues, centrosome maturation begins to occur with the new daughter centrosome recruiting associated proteins. This process does not fully complete until the following cell cycle, resulting in a mother centrosome that contains the mother centriole, and a daughter centrosome that contains the previously daughter centriole (Nigg and Stearns, 2011). Up through G2 in the cell cycle both pairs of centrioles continue to act as one centrosome to allow for proper MT organization. Once the cell enters mitosis, centrosome separation occurs to allow for the formation of separate spindle poles. Each daughter cell then inherits one functional centrosome. Development of Supernumerary Centrosomes When errors occur in the centrosome cycle, supernumerary centrosomes can develop in cells. There are four accepted models for the origin of supernumerary centrosomes (Nigg, 2002): 1) Overduplication of centrosomes can occur if there is a disconnect between centrosome duplication and chromosomal duplication during S phase of the cell cycle. Chromosomal damage can result in a halt of progression
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]
  • 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]
  • 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]
  • Supplemental Information Proximity Interactions Among Centrosome
    Current Biology, Volume 24 Supplemental Information Proximity Interactions among Centrosome Components Identify Regulators of Centriole Duplication Elif Nur Firat-Karalar, Navin Rauniyar, John R. Yates III, and Tim Stearns Figure S1 A Myc Streptavidin -tubulin Merge Myc Streptavidin -tubulin Merge BirA*-PLK4 BirA*-CEP63 BirA*- CEP192 BirA*- CEP152 - BirA*-CCDC67 BirA* CEP152 CPAP BirA*- B C Streptavidin PCM1 Merge Myc-BirA* -CEP63 PCM1 -tubulin Merge BirA*- CEP63 DMSO - BirA* CEP63 nocodazole BirA*- CCDC67 Figure S2 A GFP – + – + GFP-CEP152 + – + – Myc-CDK5RAP2 + + + + (225 kDa) Myc-CDK5RAP2 (216 kDa) GFP-CEP152 (27 kDa) GFP Input (5%) IP: GFP B GFP-CEP152 truncation proteins Inputs (5%) IP: GFP kDa 1-7481-10441-1290218-1654749-16541045-16541-7481-10441-1290218-1654749-16541045-1654 250- Myc-CDK5RAP2 150- 150- 100- 75- GFP-CEP152 Figure S3 A B CEP63 – – + – – + GFP CCDC14 KIAA0753 Centrosome + – – + – – GFP-CCDC14 CEP152 binding binding binding targeting – + – – + – GFP-KIAA0753 GFP-KIAA0753 (140 kDa) 1-496 N M C 150- 100- GFP-CCDC14 (115 kDa) 1-424 N M – 136-496 M C – 50- CEP63 (63 kDa) 1-135 N – 37- GFP (27 kDa) 136-424 M – kDa 425-496 C – – Inputs (2%) IP: GFP C GFP-CEP63 truncation proteins D GFP-CEP63 truncation proteins Inputs (5%) IP: GFP Inputs (5%) IP: GFP kDa kDa 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl Myc- 150- Myc- 100- CCDC14 KIAA0753 100- 100- 75- 75- GFP- GFP- 50- CEP63 50- CEP63 37- 37- Figure S4 A siCtl
    [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]
  • CEP250/CNAP1 Polyclonal Antibody Catalog Number:14498-1-AP 21 Publications
    For Research Use Only CEP250/CNAP1 Polyclonal antibody www.ptglab.com Catalog Number:14498-1-AP 21 Publications Catalog Number: GenBank Accession Number: Purification Method: Basic Information 14498-1-AP BC001433 Antigen affinity purification Size: GeneID (NCBI): Recommended Dilutions: 150ul , Concentration: 800 μg/ml by 11190 WB 1:500-1:1000 Nanodrop and 460 μg/ml by Bradford Full Name: IP 0.5-4.0 ug for IP and 1:500-1:1000 method using BSA as the standard; centrosomal protein 250kDa for WB Source: IHC 1:50-1:500 Calculated MW: IF 1:50-1:500 Rabbit 281 kDa Isotype: Observed MW: IgG 250 kDa Immunogen Catalog Number: AG5925 Applications Tested Applications: Positive Controls: IF, IHC, IP, WB,ELISA WB : HeLa cells, Cited Applications: IP : HeLa cells, IF, WB IHC : human placenta tissue, Species Specificity: human IF : HeLa cells, Cited Species: human, mouse Note-IHC: suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0 CEP250, also known as C-Nap1, is a 250 kDa coiled-coil protein that localizes to the proximal ends of mother and Background Information daughter centrioles. It is required for centriole-centriole cohesion during interphase of the cell cycle. It dissociates from the centrosomes when parental centrioles separate at the beginning of mitosis. The protein associates with and is phosphorylated by NIMA-related kinase 2, which is also associated with the centrosome. Notable Publications Author Pubmed ID Journal Application Yuki Yoshino 32788231 J Cell Sci IF Biao Wang 33987909 EMBO Rep IF Johan Busselez 31076588 Sci Rep Storage: Storage Store at -20°C.
    [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]
  • Myopia in African Americans Is Significantly Linked to Chromosome 7P15.2-14.2
    Genetics Myopia in African Americans Is Significantly Linked to Chromosome 7p15.2-14.2 Claire L. Simpson,1,2,* Anthony M. Musolf,2,* Roberto Y. Cordero,1 Jennifer B. Cordero,1 Laura Portas,2 Federico Murgia,2 Deyana D. Lewis,2 Candace D. Middlebrooks,2 Elise B. Ciner,3 Joan E. Bailey-Wilson,1,† and Dwight Stambolian4,† 1Department of Genetics, Genomics and Informatics and Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States 2Computational and Statistical Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Baltimore, Maryland, United States 3The Pennsylvania College of Optometry at Salus University, Elkins Park, Pennsylvania, United States 4Department of Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, United States Correspondence: Joan E. PURPOSE. The purpose of this study was to perform genetic linkage analysis and associ- Bailey-Wilson, NIH/NHGRI, 333 ation analysis on exome genotyping from highly aggregated African American families Cassell Drive, Suite 1200, Baltimore, with nonpathogenic myopia. African Americans are a particularly understudied popula- MD 21131, USA; tion with respect to myopia. [email protected]. METHODS. One hundred six African American families from the Philadelphia area with a CLS and AMM contributed equally to family history of myopia were genotyped using an Illumina ExomePlus array and merged this work and should be considered co-first authors. with previous microsatellite data. Myopia was initially measured in mean spherical equiv- JEB-W and DS contributed equally alent (MSE) and converted to a binary phenotype where individuals were identified as to this work and should be affected, unaffected, or unknown.
    [Show full text]
  • 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]
  • 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]
  • Coordination of Centrosome Homeostasis and DNA Repair Is Intact in MCF-7 and Disrupted in MDA-MB 231 Breast Cancer Cells
    Published OnlineFirst April 13, 2010; DOI: 10.1158/0008-5472.CAN-09-3800 Tumor and Stem Cell Biology Cancer Research Coordination of Centrosome Homeostasis and DNA Repair Is Intact in MCF-7 and Disrupted in MDA-MB 231 Breast Cancer Cells Ilie D. Acu1, Tieju Liu1,3, Kelly Suino-Powell1,4, Steven M. Mooney1, Antonino B. D'Assoro1, Nicholas Rowland1, Alysson R. Muotri5, Ricardo G. Correa6, Yun Niu3, Rajiv Kumar1,2, and Jeffrey L. Salisbury1 Abstract When cells encounter substantial DNA damage, critical cell cycle events are halted while DNA repair mechanisms are activated to restore genome integrity. Genomic integrity also depends on proper assembly and function of the bipolar mitotic spindle, which is required for equal chromosome segregation. Failure to execute either of these processes leads to genomic instability, aging, and cancer. Here, we show that following DNA damage in the breast cancer cell line MCF-7, the centrosome protein centrin2 moves from the cytoplasm and accumulates in the nucleus in a xeroderma pigmentosum complementation group C protein (XPC)– dependent manner, reducing the available cytoplasmic pool of this key centriole protein and preventing cen- trosome amplification. MDA-MB 231 cells do not express XPC and fail to move centrin into the nucleus following DNA damage. Reintroduction of XPC expression in MDA-MB 231 cells rescues nuclear centrin2 sequestration and reestablishes control against centrosome amplification, regardless of mutant p53 status. Importantly, the capacity to repair DNA damage was also dependent on the availability of centrin2 in the nucleus. These observations show that centrin and XPC cooperate in a reciprocal mechanism to coordinate centrosome homeostasis and DNA repair and suggest that this process may provide a tractable target to develop treatments to slow progression of cancer and aging.
    [Show full text]
  • Specialized Cilia in Mammalian Sensory Systems
    Cells 2015, 4, 500-519; doi:10.3390/cells4030500 OPEN ACCESS cells ISSN 2073-4409 www.mdpi.com/journal/cells Review Specialized Cilia in Mammalian Sensory Systems Nathalie Falk, Marlene Lösl, Nadja Schröder and Andreas Gießl * Department of Biology, Animal Physiology, University of Erlangen-Nuremberg, 91058 Erlangen, Germany; E-Mails: [email protected] (N.F.); [email protected] (M.L.); [email protected] (A.G.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +49-9131-85-28055; Fax: +49-9131-85-28060. Academic Editors: Gang Dong and William Tsang Received: 18 May 2015 / Accepted: 9 September 2015 / Published: 11 September 2015 Abstract: Cilia and flagella are highly conserved and important microtubule-based organelles that project from the surface of eukaryotic cells and act as antennae to sense extracellular signals. Moreover, cilia have emerged as key players in numerous physiological, developmental, and sensory processes such as hearing, olfaction, and photoreception. Genetic defects in ciliary proteins responsible for cilia formation, maintenance, or function underlie a wide array of human diseases like deafness, anosmia, and retinal degeneration in sensory systems. Impairment of more than one sensory organ results in numerous syndromic ciliary disorders like the autosomal recessive genetic diseases Bardet-Biedl and Usher syndrome. Here we describe the structure and distinct functional roles of cilia in sensory organs like the inner ear, the olfactory epithelium, and the retina of the mouse. The spectrum of ciliary function in fundamental cellular processes highlights the importance of elucidating ciliopathy-related proteins in order to find novel potential therapies.
    [Show full text]