DOCSLIB.ORG

Mitochondrial and Mitochondrial DNA Inheritance Checkpoints in The

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mitochondrial and Mitochondrial DNA Inheritance Checkpoints in The MitochondrialandMitochondrialDNAInheritance CheckpointsintheBuddingYeast, Saccharomyces cerevisiae DavidGarryCrider Submittedinpartialfulfillmentoftherequirementsforthe degreeofDoctorofPhilosophyintheGraduateSchoolof ArtsandSciences COLUMBIAUNIVERSITY 2012 ©2011 DavidGarryCrider Allrightsreserved ABSTRACT MitochondrialandMitochondrialDNAInheritance CheckpointsintheBuddingYeast, Saccharomycescerevisiae DavidGarryCrider This dissertation analyzes the importance of mitochondria and mitochondrial DNA in Saccharomyces cerevisiae during cell division . Movement and positional control of mitochondria and other organelles are coordinated with cell cycle progression in the buddingyeast,Saccharomycescerevisiae.Recentstudieshaverevealedacheckpoint thatinhibitscytokinesiswhenthereareseveredefectsinmitochondrialinheritance.An establishedcheckpointsignalingpathway,themitoticexitnetwork(MEN),participatesin this process. Here, we describe mitochondrial motility during inheritance in budding yeast, emerging evidence for mitochondrial quality control during inheritance, and organelleinheritancecheckpointsformitochondriaandotherorganelles. TABLE OF CONTENTS CHAPTER1................................................................................................1 INTRODUCTION........................................................................................1 MITOCHONDRIA....................................................................................................................2 CELLCYCLE..........................................................................................................................2 CHECKPOINTS......................................................................................................................5 MITOCHONDRIALINHERITANCE.......................................................................................10 QUALITYCONTROLDURINGMITOCHONDRIALINHERITANCE......................................11 MITOTICEXITNETWORKFUNCTIONINTHEMITOCHONDRIALINHERITANCE CHECKPOINT......................................................................................................................16 CHAPTER2 ........................................................................................................................20 MITOCHONDRIALINHERITANCEISREQUIREDFORMEN- REGULATEDCYTOKINESISINBUDDINGYEAST ..........................................20 SUMMARY...........................................................................................................................21 RESULTSANDDISCUSSION..............................................................................................22 Mutationsthatinhibitmitochondrialinheritanceproducemultibuddedcellsinbuddingyeast...........22 mdm10 ∆cellsexhibitdefectsincontractileringclosure....................................................................26 RolefortheMENinregulationofcellcycleprogressioninmdm10 ∆cells........................................32 ExperimentalProcedures...................................................................................................................38 Yeaststrains,plasmids,andgrowthconditions:................................................................................42 Yeaststrainsusedforthisstudy........................................................................................................43 CHAPTER3..............................................................................................46 MtDNAINHERITANCECHECKPOINT.....................................................46 BACKGROUND....................................................................................................................47 OTHERPROTEINSIMPLICATEDINmtDNAINHERITANCE..............................................48 mtDNAMUTATIONS:rho 0ANDrho -CELLS........................................................................49 Rad53ANDTHEDNADAMAGECHECKPOINT..................................................................51 LINKSBETWEENRad53ANDmtDNA................................................................................54 RESULTS.............................................................................................................................56 LOSSOFmtDNAINDUCESAG1ARRESTINCELLCYCLEPROGRESSION............................56 i THEG1TOSPROGRESSIONDEFECTOBSERVEDINCELLSLACKINGmtDNAISNOTDUE TOLOSSOFMITOCHONDRIALRESPIRATORYACTIVITYORENERGYPRODUCTION..........58 THEDEFECTINCELLCYCLEPROGRESSIONOBSERVEDINrho 0CELLSISDUETOLOSSOF DNAINMITOCHONDRIAANDNOTGENESENCODEDBYTHATDNA.......................................61 ROLEFORAKNOWNCHECKPOINTPROTEIN(Rad53)INREGULATIONOFCELLCYCLE PROGRESSIONINCELLSLACKINGmtDNA..................................................................................62 DISCUSSION.......................................................................................................................65 EXPERIMENTALPROCEDURES........................................................................................68 Yeaststrains,plasmids,andgrowthconditions:................................................................................68 CHAPTER4..............................................................................................72 DISCUSSION............................................................................................72 DISCUSSION.......................................................................................................................73 SUMMARYOFPROJECT1:................................................................................................74 FUTUREEXPERIMENTS:....................................................................................................75 SUMMARYOFPROJECT2:................................................................................................77 ROLEFORDNAPol γASASENSORFORTHEmtDNAINHERITANCECHECKPOINT:...79 HOWISTHESIGNALTHATmtDNALOSSTRANSMITTEDFROMMITOCHONDRIATO THENUCLEUS?..................................................................................................................83 POSSIBLEROLEFORPROHIBITINSINTHEmtDNAINHERITANCECHECKPOINT.......85 HOWMYSTUDIESMAYCONTRIBUTETOOURUNDERSTANDINGOF MITOCHONDRIALDISEASES.............................................................................................87 BIBLIOGRAPHY.......................................................................................90 ii ACKNOWLEDGEMENTS IwouldliketothankLizaPonforhermentorshipoverthepast3years.Thelab environmentshecreatedgiveseverygraduatestudenttheopportunityto succeed.Shesettheperfectbalancebetweenscientificcuriosityandreality;for whentofocusandfinishupthestory.Icannotthankherenoughforher dedicationandguidance. ThePonlabwouldnotfunctionasefficiently,oraswellasitdoes,withoutIstvan Boldogh.Heisthebackboneofthelab.Hehassuchawealthofknowledge,and suchaselflesspersonalitythathewilldropeverythingandhelpyouwheneverhe can.Iamsogratefulforallourconversationsandthetimewespenttogether.He trulymadethelabafunplace. IwouldliketothankTomLipkinfortakingthetimetograbacupofcoffeeand encouragemetojointhePonlab.TheresaSwaynemightnotrealizethatshewas thefirstpersonthatItalkedtowhenIcametoColumbia(duringmyorientation) andI’mhonoredtobeabletothankheronmylastdayatColumbia.ToJose “Ricky”RicardoMcFalineFigueroa(pickanameman!)whoislikeanolder,I meanyounger,brotherthatIneverhad,thankyou.Wediscussedverylittle scienceandforthatIcreditmycurrentsanity.ToLuisGarcia,whoIhavenever met,butwouldnothavehadsuchanexcitingprojectifitwasn’tforhisdiscovery andpreviouswork. iii Iwouldliketothankmyfirstscientificmentor,KennethBoheler,whoencouraged out-of-the-boxthinkingandmoldedmeintoascientist.Tothisdaymymost excitingexperimentwaswatchingmystemcellderivedcardiomyocytesbeaton thedish.WhowouldhavethoughtthatsingleexperimentwouldbethereasonI metmywife.I’dliketothankthoselittlecardiomyocytesforgrabbingmywife’s attentionandgivingmethechancetomeether,fallinlove,andgetmarried…my nextmostexcitingexperimentsareyettocome. Myfamilyhasprovidedunconditionalsupportformethroughoutthelastdecade asIwouldfinishonedegreeandstartthenext.Theyneverquestionedmy dedicationandalwaysunderstoodandacceptedthesacrificethatittooktoreach mygoal.Icannevergetthoselostholidaysandtimespentawayfromthemback butIhopethesacrificeisjustifiedintheend. Tomypap,thebestself-taughtscientistthatIknow. Everyonementionedhadahugeroleintogettingmetothisdaybutnoone deservesmorecreditthanmywifeCheryl.SheisthereasonIcamebackto graduateschoolmorefocusedanddeterminedthanever.Icannotthankher enoughforherpatienceandunderstandingwhenIhadtoworkeveryweekend andstayinlablatebutalsoforthosedayswheresheputherfootdownand physicallydraggedmeoutoflab.SheknewwhenIwasburningmyselfoutand whentotellmetogetmyassoffthecouchandfinish.Ican’tdescribehowmuch youmeantomeandIcouldn’thavecompletedthisjourneywithoutyouandI cannotwaittostartthenextjourneywithyou. iv 1 CHAPTER1 INTRODUCTION 2 MITOCHONDRIA Mitochondria are essential organelles that perform fundamental cellular functions includingaerobicmetabolicfattyacidoxidation,aminoacidmetabolism,apoptosisand biosynthesis of many cellular metabolites. Mitochondria contain their own DNA (mtDNA), which encodes respiratory chain components and tRNAs necessary for synthesis of mtDNA-encoded respiratory chain components, and cannot be made de novo. Saccharomyces cerevisiae has become an ideal model system for studying mitochondrialfunctionandinheritance,processesthatarerequiredforcellsurvival.The vital process of replicating and transferring ‘fit’ mitochondria
Load more

Recommended publications
  • Chloroplast Transit Peptides: Structure, Function and Evolution
    reviews Chloroplast transit Although the first demonstration of precursor trans- port into chloroplasts was shown over two decades peptides: structure, ago3,4, only now is this area of cell biology becom- ing well understood. Many excellent reviews have been published recently on the evolution of plas- function and tids5, the evolution of organelle genomes6, the mechanism of gene transfer from organelles to the nucleus7 and the mechanism of protein import into evolution chloroplasts8,9. Proteins destined to plastids and other organ- elles share in common the requirement for ‘new’ Barry D. Bruce sequence information to facilitate their correct trafficking within the cell. Although in most cases this information resides in a cleavable, N-terminal sequence often collectively referred to as signal It is thought that two to three thousand different proteins are sequence, the different organelle-targeting se- targeted to the chloroplast, and the ‘transit peptides’ that act as quences have distinct properties and names: ‘signal peptides’ for the endoplasmic reticulum, chloroplast targeting sequences are probably the largest class of ‘presequences’ for the mitochondria and ‘transit peptides’ for chloroplasts and other plastids. This targeting sequences in plants. At a primary structural level, transit review focuses on recent progress in dissecting peptide sequences are highly divergent in length, composition and the role of the stromal-targeting domain of chloro- plast transit peptides. I will consider briefly the organization. An emerging concept suggests that transit peptides multitude of distinct functions that transit peptides contain multiple domains that provide either distinct or overlapping perform, provide an update on the limited struc- tural information of a number of transit peptides functions.
    [Show full text]
  • Asymmetric Distribution of Glucose Transporter Mrna Provides Growth Advantage
    bioRxiv preprint doi: https://doi.org/10.1101/380279; this version posted July 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Asymmetric Distribution of Glucose Transporter mRNA Provides Growth Advantage Timo Stahl1, Stefan Hümmer1,2, Nikolaus Ehrenfeuchter1, Geoffrey Fucile3, and Anne Spang1 1Biozentrum, University of Basel, 4056 Basel, Switzerland 2current affiliation: Translational Molecular Pathology, Vall d'Hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona and Spanish Biomedical Research Network Centre in Oncology (CIBERONC), Spain 3SIB Swiss Institute of Bioinformatics, sciCORE Computing Center, University of Basel, 4056 Basel, Switzerland Address of Correspondence: Anne Spang Biozentrum University of Basel Klingelbergstrasse 70 CH-4056 Basel Switzerland Email: [email protected] Phone: +41 61 207 2380 Running title: PKA asymmetrically localizes HXT2 mRNA 1 bioRxiv preprint doi: https://doi.org/10.1101/380279; this version posted July 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract (175 words) Asymmetric localization of mRNA is important for cell fate decisions in eukaryotes and provides the means for localized protein synthesis in a variety of cell types. Here we show that hexose transporter mRNAs are retained in the mother cell of S. cerevisiae until metaphase-anaphase transition (MAT) and then are released into the bud.
    [Show full text]
  • Many Routes Lead to the Pole Nidulans and the Fission Yeast Schizo.Mc­ Charomyces Pombe, Genes Required for the Caroline E
    NEWS AND VIEWS remaining material. One intriguing pro­ planets and under the sea, and especially One of the most surprising has emerged posal (R. F. Scott, California Institute of of the artificially triggered Soviet geo­ from the study of genes concerned with Technology) was that a 10-m diameter, technical landslides, will shed new light another process associated with the pole 1,000 g geotechnical centrifuge would on this vexing question. It is clear that body, namely, the movement of nuclei permit model experiments on dynam­ Western geoscientists have much to gain towards one another during karyogamy. ically scaled analogues of natural large from cooperation with our Soviet counter­ One of these, KARI, encodes a pole-body landslides. parts in the new spirit of glasnost. D component of unknown function 111 but In spite of the flood of new data on giant another, KAR3, has been shown to landslides, the fundamental cause of their H. J. Me/ash is in the Lunar and Planetary encode a homologue of the microtubule 11 mobility is still far from understood. It is Laboratory and Department of Geosciences, motor protein kinesin • This colocalizes hoped that the new opportunities for the University of Arizona, Tucson, Arizona 85721, with the KARI product at the outer sur­ study of their deposits both on other USA. face of the pole body during conjugation CELL BIOLOGY-------------------- but resides at the inner face during mitosis (M. Rose, personal communication). In the filamentous fungus Aspergillus Many routes lead to the pole nidulans and the fission yeast Schizo.mc­ charomyces pombe, genes required for the Caroline E.
    [Show full text]
  • Mutant Membrane Protein of the Budding Yeast Spindle Pole Body Is Targeted to the Endoplasmic Reticulum Degradation Pathway
    Copyright 2002 by the Genetics Society of America Mutant Membrane Protein of the Budding Yeast Spindle Pole Body Is Targeted to the Endoplasmic Reticulum Degradation Pathway Susan McBratney and Mark Winey1 Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347 Manuscript received June 5, 2001 Accepted for publication June 3, 2002 ABSTRACT Mutation of either the yeast MPS2 or the NDC1 gene leads to identical spindle pole body (SPB) duplication defects: The newly formed SPB is improperly inserted into the nuclear envelope (NE), preventing the cell from forming a bipolar mitotic spindle. We have previously shown that both MPS2 and NDC1 encode integral membrane proteins localized at the SPB. Here we show that CUE1, previously known to have a role in coupling ubiquitin conjugation to ER degradation, is an unusual dosage suppressor of mutations in MPS2 and NDC1. Cue1p has been shown to recruit the soluble ubiquitin-conjugating enzyme, Ubc7p, to the cytoplasmic face of the ER membrane where it can ubiquitinate its substrates and target them for degradation by the proteasome. Both mps2-1 and ndc1-1 are also suppressed by disruption of UBC7 or its partner, UBC6. The Mps2-1p mutant protein level is markedly reduced compared to wild-type Mps2p, and deletion of CUE1 restores the level of Mps2-1p to nearly wild-type levels. Our data indicate that Mps2p may be targeted for degradation by the ER quality control pathway. N the budding yeast Saccharomyces cerevisiae, the spin- martin 1999; O’Toole et al. 1999). It was originally I dle pole body (SPB) functions as the sole microtu- proposed that Mps2p and Ndc1p function to insert the bule-organizing center (Byers et al.
    [Show full text]
  • SPC72: a Spindle Pole Component Required for Spindle Orientation in the Yeast Saccharomyces Cerevisiae
    Journal of Cell Science 111, 2809-2818 (1998) 2809 Printed in Great Britain © The Company of Biologists Limited 1998 JCS3813 SPC72: a spindle pole component required for spindle orientation in the yeast Saccharomyces cerevisiae Sylvie Souès* and Ian R. Adams Laboratory of Molecular Biology, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, UK *Author for correspondence at present address: Institut de Génétique et de Microbiologie, Bât. 400, Université de Paris-Sud XI, 91 405 Orsay Cédex, France (e-mail: [email protected]) Accepted 7 July; published on WWW 27 August 1998 SUMMARY The monoclonal antibody 78H6 recognises an 85 kDa mating capability. Precisely, deletion of SPC72 resulted in component of the yeast spindle pole body. Here we identify a decreased number of astral microtubules: early in the cell and characterise this component as Spc72p, the product of cycle only few were detectable, and these were unattached YAL047C. The sequence of SPC72 contains potential coiled- to the spindle pole body in small-budded cells. Later in the coil domains; its overexpression induced formation of large cell cycle few, if any, remained, and they were unable to polymers that were strictly localised at the outer plaque align the spindle properly. We conclude that Spc72p is not and at the bridge of the spindle pole body. Immunoelectron absolutely required for nucleation per se, but is needed for microscopy confirmed that Spc72p was a component of normal abundance and stability of astral microtubules. these polymers. SPC72 was found to be non-essential for cell growth, but its deletion resulted in abnormal spindle Key words: Yeast, Spindle Pole Body, Astral microtubule, positioning, aberrant nuclear migration and defective Microtubule dynamic, Nuclear migration, Mating INTRODUCTION fails to orient the spindle towards the bud neck, with the consequence that nuclei fail to segregate appropriately (Palmer Microtubules play a key role in mitosis and mating of yeast.
    [Show full text]
  • Osu1343763753.Pdf (14.8
    Targeting of Peripherally Associated Proteins to the Inner Nuclear Membrane in Saccharomyces cerevisiae: The Role of Essential Proteins DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Greetchen M. Díaz Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2012 Dissertation Committee: Anita K. Hopper, Advisor Stephen Osmani Mark Parthun Jian-Qiu Wu Copyright by Greetchen M. Díaz 2012 Abstract The nuclear envelope (NE) is composed of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) which is contiguous with the endoplasmic reticulum (ER). The appropriate location of NE proteins is important in cells. Integral INM proteins are proposed to be synthesized at the ER and then translocated through the nuclear pore complex (NPC). In contrast, peripherally associated INM proteins are proposed to follow a targeting mechanism to the nucleus that is similar to nucleoplasmic proteins. Our research aims to understand the mechanism of targeting of peripherally associated proteins to the INM. We employed yeast as a genetic model and the tRNA modification enzyme, Trm1-II, as a reporter. We screened a collection of temperature sensitive (ts) mutants for defects in galactose-inducible Trm1-II-GFP (Gal-Trm1-II-GFP) INM localization. We found that the majority (46%) of the ts mutations affecting Gal- Trm1-II-GFP localization were in genes that encode proteins involved in ER-Golgi homeostasis. Interestingly, about 35% of the mutated essential genes encode components of the Spindle Pole Body (SPB). In the SPB ts mutants, at the non-permissive temperature, Gal-Trm1-II-GFP accumulates as a spot that localizes to the ER, rather than being evenly distributed around the entire INM as in wild-type cells.
    [Show full text]
  • Cytoskeleton: Anatomy of an Organizing Center Laura G
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE R754 Dispatch provided by Elsevier - Publisher Connector Cytoskeleton: Anatomy of an organizing center Laura G. Marschall and Tim Stearns One component of the yeast spindle pole body, Spc42p, SPB are duplicated each cell cycle, such that a new has been found to form a crystalline array within one of organelle is created next to the existing one. Unlike DNA the central layers of the structure; the Spc42p crystal replication, where the structure of the molecule immedi- might provide a scaffold around which the spindle pole ately suggests a templated mechanism of replication, body is assembled, and could be involved in regulating there is no obvious template in either the centrosome or the size of the spindle pole body. SPB. Two recent papers suggest that the SPB might be assembled from the inside out, starting with a central Address: Department of Biological Sciences, Stanford University, Stanford, California 94305, USA. crystalline array of a single protein [1], to which other components are added [2]. Current Biology 1997, 7:R754–R756 http://biomednet.com/elecref/09609822007R0754 Early studies showed that, when viewed by thin-section © Current Biology Ltd ISSN 0960-9822 electron microscopy, the SBP appears to consist of three layers (reviewed in [3]). The central plaque is embedded Most cells in your body have an extensive microtubule in the nuclear envelope, the inner plaque is on the nuclear network that is busily transporting organelles and vesicles side of the central plaque, and the outer plaque is on the from one place to another, and separating chromosomes in cytoplasmic side of the central plaque.
    [Show full text]
  • Atspc98p and Plant Microtubule Nucleation 2425
    Research Article 2423 The plant Spc98p homologue colocalizes with γ-tubulin at microtubule nucleation sites and is required for microtubule nucleation Mathieu Erhardt1, Virginie Stoppin-Mellet1,*, Sarah Campagne1, Jean Canaday1, Jérôme Mutterer1, Tanja Fabian2, Margret Sauter1,2, Thierry Muller1, Christine Peter1, Anne-Marie Lambert1 and Anne-Catherine Schmit1,‡ 1Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique UPR 2357, Université Louis Pasteur, 12 rue du Général Zimmer F-67084, Strasbourg Cedex, France 2Institut für Allgemeine Botanik, Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany *Present address: Laboratoire de Physiologie Cellulaire Végétale, UMR 5019 CEA/CNRS/UJF, CEA Grenoble, 17 Avenue des Martyrs 38054 Grenoble cedex 9, France ‡Author for correspondence (e-mail: [email protected]) Accepted 13 March 2002 Journal of Cell Science 115, 2423-2431 (2002) © The Company of Biologists Ltd Summary The molecular basis of microtubule nucleation is still not tobacco nuclei and in living cells. AtSpc98p-GFP also known in higher plant cells. This process is better localizes at the cell cortex. Spc98p is not associated with γ- understood in yeast and animals cells. In the yeast spindle tubulin along microtubules. These data suggest that pole body and the centrosome in animal cells, γ-tubulin multiple microtubule-nucleating sites are active in plant small complexes and γ-tubulin ring complexes, respectively, cells. Microtubule nucleation involving Spc98p-containing nucleate all microtubules. In addition to γ-tubulin, Spc98p γ-tubulin complexes could then be conserved among all or its homologues plays an essential role. We report here eukaryotes, despite differences in structure and spatial the characterization of rice and Arabidopsis homologues of distribution of microtubule organizing centers.
    [Show full text]
  • The JNM1 Gene in the Yeast Saccharomyces Cerevisiae Is Required for Nuclear Migration and Spindle Orientation During the Mitotic Cell Cycle John N
    The JNM1 Gene in the Yeast Saccharomyces cerevisiae Is Required for Nuclear Migration and Spindle Orientation During the Mitotic Cell Cycle John N. McMillan and Kelly Tatchell Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27695 Abstract. JNM1, a novel gene on chromosome XIII in during mitosis, as assayed by the rate of chromosome the yeast Saccharomyces cerevisiae, is required for loss, or nuclear migration during conjugation, as as- proper nuclear migration, jnml null mutants have a sayed by the rate of mating and cytoduction. The temperature-dependent defect in nuclear migration and phenotype of a jnml mutant is strikingly similar to an accompanying alteration in astral microtubules. At that for mutants in the dynein heavy chain gene 30°C, a significant proportion of the mitotic spindles (Eshel, D., L. A. Urrestarazu, S. Vissers, J.-C. is not properly located at the neck between the mother Jauniaux, J. C. van Vliet-Reedijk, R. J. Plants, and cell and the bud. This defect is more severe at low I. R. Gibbons. 1993. Proc. Natl. Acad. Sci. USA. temperature. At ll°C, 60% of the cells accumulate 90:11172-11176; Li, Y. Yo, E. Yeh, T. Hays, and K. with large buds, most of which have two DAPI stain- Bloom. 1993. Proc. Natl. Acad. Sci. USA. 90:10096- ing regions in the mother cell. Although mitosis is 10100). The JNM1 gene product is predicted to en- delayed and nuclear migration is defective in jnml mu- code a 44-kD protein containing three coiled coil do- tants, we rarely observe more than two nuclei in a mains.
    [Show full text]
  • Impact on Spindle Pole Body Separation and Chromosome Capture Beryl Augustine, Cheen Fei Chin and Foong May Yeong*
    © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs211425. doi:10.1242/jcs.211425 RESEARCH ARTICLE Role of Kip2 during early mitosis – impact on spindle pole body separation and chromosome capture Beryl Augustine, Cheen Fei Chin and Foong May Yeong* ABSTRACT MTs (cMTs), which are present outside the nucleus. cMTs, Mitotic spindle dynamics are regulated during the cell cycle by otherwise known as astral MTs (aMTs), are an integral microtubule motor proteins. In Saccharomyces cerevisiae, one such component of the mitotic spindle and play an important role in protein is Kip2p, a plus-end motor that regulates the polymerization nuclear migration from the mother to daughter cell and in spindle and stability of cytoplasmic microtubules (cMTs). Kip2p levels are positioning (Shaw et al., 1997). regulated during the cell cycle, and its overexpression leads to the The dynamics of the mitotic spindle including its assembly, formation of hyper-elongated cMTs. To investigate the significance of disassembly and positioning, are mediated by force generation from varying Kip2p levels during the cell cycle and the hyper-elongated polymerization and depolymerization of MTs themselves, and from cMTs, we overexpressed KIP2 in the G1 phase and examined the MT-associated motor proteins (McCarthy and Goldstein, 2006). effects on the separation of spindle pole bodies (SPBs) and MTs undergo assembly and disassembly to generate pushing and chromosome segregation. Our results show that failure to regulate pulling forces, respectively (Dogterom et al., 2005). In vitro the cMT lengths during G1-S phase prevents the separation of SPBs. experiments have shown that MT assembly or disassembly by This, in turn, affects chromosome capture and leads to the activation themselves can create forces strong enough to transport organelles of spindle assembly checkpoint (SAC) and causes mitotic arrest.
    [Show full text]
  • Hsp110 Is Required for Spindle Length Control
    Published August 20, 2012 JCB: Article Hsp110 is required for spindle length control Taras Makhnevych,1 Philip Wong,4 Oxana Pogoutse,2,3 Franco J. Vizeacoumar,2,3 Jack F. Greenblatt,2,3 Andrew Emili,2,3 and Walid A. Houry1 1Department of Biochemistry, 2Department of Molecular Genetics, and 3Banting and Best Department of Medical Research, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 1A8, Canada 4Institute of Bioinformatics and Systems Biology, Helmholtz Center Munich, German Research Center for Environmental Health, D-85764 Neuherberg, Germany ystematic affinity purification combined with mass of the widely conserved kinesin-5 motor Cin8. The role spectrometry analysis of N- and C-tagged cytoplas- of Hsp110 Sse1 as a nucleotide exchange factor for the S mic Hsp70/Hsp110 chaperones was used to iden- Hsp70 chaperones Ssa1/Ssa2 was found to be required tify new roles of Hsp70/Hsp110 in the cell. This allowed for maintaining the proper distribution of kinesin-5 motors the mapping of a chaperone–protein network consisting of within the spindle, which was subsequently required for 1,227 unique interactions between the 9 chaperones and bipolar spindle assembly in S phase. These data suggest Downloaded from 473 proteins and highlighted roles for Hsp70/Hsp110 a model whereby the Hsp70–Hsp110 chaperone complex in 14 broad biological processes. Using this information, antagonizes Cin8 plus-end motility and prevents prema- we uncovered an essential role for Hsp110 in spindle ture spindle elongation in S phase. assembly and, more specifically, in modulating the activity jcb.rupress.org Introduction The Hsp70 family of chaperones is a multifunctional group of function (Huang et al., 2005).
    [Show full text]
  • Preparation of Yeast Nuclei and Spindle Pole Bodies (Michael Rout and John Kilmartin)
    Rout Lab Protocol Last Modified by Michael Rout 3/15/07 Preparation of Yeast Nuclei and Spindle Pole Bodies (Michael Rout and John Kilmartin) I. Introduction Spindle pole bodies (SPBs) are the sole microtubule organizing centers of budding yeast cells. SPBs are embedded in the nuclear envelope which remains intact during mitosis thus spindles are intranuclear. SPBs can be enriched six hundred fold and in high yield from Saccharomyces uvarum (Rout and Kilmartin, 1990). The procedure involves preparation of nuclei by a modification of an existing method (Rozijn and Tonino, 1964). The nuclei are then lysed and extracted to free the SPBs from the nuclear envelope, followed by two gradient steps to separate the SPBs from other nuclear components. These SPBs, which are about 10% pure, have been used to prepare mAbs and thereby identify components of the SPB and spindle (Rout and Kilmartin, 1990, 1991). II. Materials and Instrumentation All catalogue numbers are in brackets. Anti foam B (A5757), sorbitol (S 1876), PVP-40 (PVP-40), pepstatin (P 4265), PMSF (P 7626), digitonin (D 1407), DNase I (DN-EP), RNase A (R 5503), GTP (G 5884), bis-Tris (B 9754), EGTA (E 4378) were obtained from Sigma, Poole, Dorset, U.K. (Fax 0800 378785). Ficoll-400 (17-0400-01), Percoll (17-0891-01) were from Pharmacia, Uppsala, Sweden. Triton X-100 (30632), glucose (10117) and dimethyl sulphoxide (DMSO, 10323) were obtained from BDH, Poole, England (Fax 0202 738299) and sucrose (5503UA) from GIBCO BRL, Uxbridge, Middlesex, U. K. (Fax 0895 53159). Yeast extract (1896) was obtained from Beta Lab, East Molesey, U.K., and bactopeptone (0118-08-1) from Difco, Detroit, MI, USA.
    [Show full text]