DOCSLIB.ORG

Role of Aurora B-Mediated Phosphorylation During Mitosis and Interphase Carmen Taveras Submitted in Partial Fulfillment Of

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Role of Aurora B-Mediated Phosphorylation During Mitosis and Interphase Carmen Taveras Submitted in Partial Fulfillment Of Role of Aurora B-mediated phosphorylation during mitosis and interphase Carmen Taveras Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Science COLUMBIA UNIVERSITY 2017 © 2017 Carmen Taveras All rights reversed ABSTRACT Role of Aurora B-mediated phosphorylation during mitosis and interphase Carmen Taveras Accurate chromosome segregation requires a spindle apparatus composed of microtubules that arise from the spindle to attach to the kinetochore, a protein complex assembled at the centromere of each chromosome. Failure to segregate chromosomes accurately may lead to lethal early developmental defects and tumorigenesis. To achieve proper kinetochore binding to microtubules, mammalian cells have evolved elaborate mechanisms to correct attachment errors and stabilize correct ones. Current models suggest that tension between kinetochore pairs (inter- kinetochore stretch) and tension at the kinetochore (intra-kinetochore stretch) produces a spatial separation of Aurora B kinase from kinetochore-associated and microtubule-binding substrates, subsequently reducing their phosphorylations and increasing their microtubule affinity. However, the tension-based models do not explain how the initial microtubule binding at unattached kinetochores occurs, where there is no tension and kinetochore-associated substrates are highly phosphorylated and, hence unable to bind to microtubules. Therefore, there must be a mechanism that explains how the phosphorylation of kinetochore substrates by Aurora B is reduced in the absence of tension. In the first part of this thesis, I examine the structural features of the coiled-coil domain of the kinetochore-associated kinesin motor protein, CENP-E. Using Single-Molecule High- Resolution Colocalization (SHREC) microscopy analysis of kinetochore-associated CENP-E, I show that CENP-E undergoes structural rearrangements prior to and after tension generation at the kinetochore. Chemical inhibition of the motor motility or genetic perturbations of the coiled-coil domain of CENP-E increases Aurora B-mediated Ndc80 phosphorylation in a tension-independent manner. Importantly, metaphase chromosome misalignment caused by inhibition of CENP-E can be rescued by chemical inhibition of Aurora B kinase. Therefore, CENP-E regulates the initial kinetochore binding to microtubules and the stabilization of kinetochore-microtubule attachments. Formin-dependent actin assembly is known to play a role in multiple processes, including cytokinesis, filopodia formation, cell polarity, and cell adhesion. Thus, formin malfunction is directly linked to various pathologies, including defects in cell migration and tumor suppression. Although the role of formins in actin polymerization has been well described, the mechanistic processes that regulate the actin assembly function of formins remain poorly understood, especially the interplay among the various sub-families of formins and how they are spatiotemporally regulated. In the second part of this thesis, I show that Aurora B-mediated phosphorylation of the formin, mDia3 regulates actin assembly. Previous studies identified two Aurora B phosphorylation sites in the FH2 domain of mDia3. To this end, phosphomimetic and non-phosphorylatable mutants of a constitutively active form of mDia3 were designed to test whether phosphorylation by Aurora B regulates actin assembly. Using an in vitro actin polymerization kinetic assay and expression of fluorescently-tagged constitutively active mDia3 in cells, I show that phosphorylation of mDia3 by Aurora B induces the actin assembly function of mDia3. Furthermore, using a phospho-specific antibody, I show that mDia3 is phosphorylated by Aurora B. Live-cell analysis shows that perturbations of these phosphorylation sites affect cell migration and cell spreading. Therefore, I illustrate a novel regulatory mechanism for the actin assembly function of mDia3 that is dependent on Aurora B kinase activity. TABLE OF CONTENTS List of figures iii Acknowledgements v Chapter 1: Introduction.................................................................................................................1 Part 1: Stabilization of kinetochore-microtubule attachments..............................................2 1. Kinetochore-mediated microtubule capture................................................................2 2. Aurora B corrects kinetochore-microtubule attachment error....................................7 3. Phosphatase recruitment to kinetochores counteract Aurora B kinase activity......... 9 4. The kinetochore phosphorylation/dephosphorylation balance is regulated by tension.......................................................................................................................11 5. CENP-E facilitates the stabilization of kinetochore-microtubule attachments.........16 6. Rationale and objectives for chapter 2......................................................................21 Part 2: Regulation of formin-mediated actin assembly.......................................................23 1. The actin cytoskeleton plays a major role in cell migration.....................................23 2. Formin-mediated actin assembly..............................................................................25 3. Aurora B-mediated phosphorylation of mDia3 regulates mDia3 function...............28 4. Rationale and objectives for chapter 3......................................................................31 Chapter 2: CENP-E regulates Aurora B kinase activity at the kinetochore..........................33 Introduction........................................................................................................................33 Materials and methods.......................................................................................................35 Results................................................................................................................................38 Discussion..........................................................................................................................64 i Chapter 3: Aurora B regulates the actin assembly function of mDia3...................................67 Introduction........................................................................................................................67 Materials and methods.......................................................................................................68 Results................................................................................................................................71 Discussion..........................................................................................................................82 Chapter 4: Discussions and future directions...........................................................................83 Part 1: CENP-E regulates Aurora B kinase activity at the kinetochore.............................83 1. Aurora B-mediated phosphorylation of outer-localized kinetochore substrates are regulated by microtubule attachment not tension.....................................................83 2. The unique structural features of CENP-E facilitate the stabilization of kinetochore- microtubule attachments before and after bi-orientation..........................................89 3. Recruitment of phosphatases to the kinetochore balances the phosphorylation of outer kinetochore-associated components................................................................99 Part 2: Aurora B regulates the actin assembly function of mDia3..................................104 1. Aurora B-mediated phosphorylation activates the actin assembly function of the FH2 domain of mDia3.................................................................104 2. Aurora B-mediated phosphorylation of mDia3 regulates the ‘crosstalk’ between the actin and the microtubule function of mDia3..................................107 3. Aurora B regulates the cytoskeleton in interphase cells......................................108 References...................................................................................................................................111 ii LIST OF FIGURES Chapter 1 Figure 1.1 Initial microtubule capture by kinetochores...................................................................4 Figure 1.2 Illustration of the tension-based models.......................................................................15 Figure 1.3. CENP-E has a unique long and flexible coiled-coil domain.......................................22 Figure 1.4 mDia3 is phosphorylated by Aurora B.........................................................................32 Chapter 2 Figure 2.1 Perturbation of the coiled-coil domain does not affect the kinetochore localization of CENP-E....................................................................................................................................41 Figure 2.2. The long and highly flexible coiled-coil domain of CENP-E is essential for its kinetochore function......................................................................................................................42 Figure 2.3. The length and flexibility of the coiled-coil domain of CENP-E mediate the folding conformation of CENP-E at the kinetochore.................................................................................44 Figure 2.4. Validation of the 3-dimensional SHREC
Load more

Recommended publications
  • Glossary - Cellbiology
    1 Glossary - Cellbiology Blotting: (Blot Analysis) Widely used biochemical technique for detecting the presence of specific macromolecules (proteins, mRNAs, or DNA sequences) in a mixture. A sample first is separated on an agarose or polyacrylamide gel usually under denaturing conditions; the separated components are transferred (blotting) to a nitrocellulose sheet, which is exposed to a radiolabeled molecule that specifically binds to the macromolecule of interest, and then subjected to autoradiography. Northern B.: mRNAs are detected with a complementary DNA; Southern B.: DNA restriction fragments are detected with complementary nucleotide sequences; Western B.: Proteins are detected by specific antibodies. Cell: The fundamental unit of living organisms. Cells are bounded by a lipid-containing plasma membrane, containing the central nucleus, and the cytoplasm. Cells are generally capable of independent reproduction. More complex cells like Eukaryotes have various compartments (organelles) where special tasks essential for the survival of the cell take place. Cytoplasm: Viscous contents of a cell that are contained within the plasma membrane but, in eukaryotic cells, outside the nucleus. The part of the cytoplasm not contained in any organelle is called the Cytosol. Cytoskeleton: (Gk. ) Three dimensional network of fibrous elements, allowing precisely regulated movements of cell parts, transport organelles, and help to maintain a cell’s shape. • Actin filament: (Microfilaments) Ubiquitous eukaryotic cytoskeletal proteins (one end is attached to the cell-cortex) of two “twisted“ actin monomers; are important in the structural support and movement of cells. Each actin filament (F-actin) consists of two strands of globular subunits (G-Actin) wrapped around each other to form a polarized unit (high ionic cytoplasm lead to the formation of AF, whereas low ion-concentration disassembles AF).
    [Show full text]
  • The Role of Model Organisms in the History of Mitosis Research
    Downloaded from http://cshperspectives.cshlp.org/ on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press The Role of Model Organisms in the History of Mitosis Research Mitsuhiro Yanagida Okinawa Institute of Science and Technology Graduate University, Okinawa 904-0495, Japan Correspondence: [email protected] Mitosis is a cell-cycle stage during which condensed chromosomes migrate to the middle of the cell and segregate into two daughter nuclei before cytokinesis (cell division) with the aid of a dynamic mitotic spindle. The history of mitosis research is quite long, commencing well before the discovery of DNA as the repository of genetic information. However, great and rapid progress has been made since the introduction of recombinant DNA technology and discovery of universal cell-cycle control. A large number of conserved eukaryotic genes required for the progression from early to late mitotic stages have been discovered, confirm- ing that DNA replication and mitosis are the two main events in the cell-division cycle. In this article, a historical overview of mitosis is given, emphasizing the importance of diverse model organisms that have been used to solve fundamental questions about mitosis. Onko Chisin—An attempt to discover new truths by checkpoint [SAC]), then metaphase (in which studying the past through scrutiny of the old. the chromosomes are aligned in the middle of cell), anaphase A (in which identical sister chro- matids comprising individual chromosomes LARGE SALAMANDER CHROMOSOMES separate and move toward opposite poles of ENABLED THE FIRST DESCRIPTION the cell), anaphase B (in which the spindle elon- OF MITOSIS gates as the chromosomes approach the poles), itosis means “thread” in Greek.
    [Show full text]
  • Mitosis Vs. Meiosis
    Mitosis vs. Meiosis In order for organisms to continue growing and/or replace cells that are dead or beyond repair, cells must replicate, or make identical copies of themselves. In order to do this and maintain the proper number of chromosomes, the cells of eukaryotes must undergo mitosis to divide up their DNA. The dividing of the DNA ensures that both the “old” cell (parent cell) and the “new” cells (daughter cells) have the same genetic makeup and both will be diploid, or containing the same number of chromosomes as the parent cell. For reproduction of an organism to occur, the original parent cell will undergo Meiosis to create 4 new daughter cells with a slightly different genetic makeup in order to ensure genetic diversity when fertilization occurs. The four daughter cells will be haploid, or containing half the number of chromosomes as the parent cell. The difference between the two processes is that mitosis occurs in non-reproductive cells, or somatic cells, and meiosis occurs in the cells that participate in sexual reproduction, or germ cells. The Somatic Cell Cycle (Mitosis) The somatic cell cycle consists of 3 phases: interphase, m phase, and cytokinesis. 1. Interphase: Interphase is considered the non-dividing phase of the cell cycle. It is not a part of the actual process of mitosis, but it readies the cell for mitosis. It is made up of 3 sub-phases: • G1 Phase: In G1, the cell is growing. In most organisms, the majority of the cell’s life span is spent in G1. • S Phase: In each human somatic cell, there are 23 pairs of chromosomes; one chromosome comes from the mother and one comes from the father.
    [Show full text]
  • The Emerging Role of Ncrnas and RNA-Binding Proteins in Mitotic Apparatus Formation
    non-coding RNA Review The Emerging Role of ncRNAs and RNA-Binding Proteins in Mitotic Apparatus Formation Kei K. Ito, Koki Watanabe and Daiju Kitagawa * Department of Physiological Chemistry, Graduate School of Pharmaceutical Science, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan; [email protected] (K.K.I.); [email protected] (K.W.) * Correspondence: [email protected] Received: 11 November 2019; Accepted: 13 March 2020; Published: 20 March 2020 Abstract: Mounting experimental evidence shows that non-coding RNAs (ncRNAs) serve a wide variety of biological functions. Recent studies suggest that a part of ncRNAs are critically important for supporting the structure of subcellular architectures. Here, we summarize the current literature demonstrating the role of ncRNAs and RNA-binding proteins in regulating the assembly of mitotic apparatus, especially focusing on centrosomes, kinetochores, and mitotic spindles. Keywords: ncRNA; centrosome; kinetochore; mitotic spindle 1. Introduction Non-coding RNAs (ncRNAs) are defined as a class of RNA molecules that are transcribed from genomic DNA, but not translated into proteins. They are mainly classified into the following two categories according to their length—small RNA (<200 nt) and long non-coding RNA (lncRNA) (>200 nt). Small RNAs include traditional RNA molecules, such as transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), PIWI-interacting RNA (piRNA), and micro RNA (miRNA), and they have been studied extensively [1]. Research on lncRNA is behind that on small RNA despite that recent transcriptome analysis has revealed that more than 120,000 lncRNAs are generated from the human genome [2–4].
    [Show full text]
  • List, Describe, Diagram, and Identify the Stages of Meiosis
    Meiosis and Sexual Life Cycles Objective # 1 In this topic we will examine a second type of cell division used by eukaryotic List, describe, diagram, and cells: meiosis. identify the stages of meiosis. In addition, we will see how the 2 types of eukaryotic cell division, mitosis and meiosis, are involved in transmitting genetic information from one generation to the next during eukaryotic life cycles. 1 2 Objective 1 Objective 1 Overview of meiosis in a cell where 2N = 6 Only diploid cells can divide by meiosis. We will examine the stages of meiosis in DNA duplication a diploid cell where 2N = 6 during interphase Meiosis involves 2 consecutive cell divisions. Since the DNA is duplicated Meiosis II only prior to the first division, the final result is 4 haploid cells: Meiosis I 3 After meiosis I the cells are haploid. 4 Objective 1, Stages of Meiosis Objective 1, Stages of Meiosis Prophase I: ¾ Chromosomes condense. Because of replication during interphase, each chromosome consists of 2 sister chromatids joined by a centromere. ¾ Synapsis – the 2 members of each homologous pair of chromosomes line up side-by-side to form a tetrad consisting of 4 chromatids: 5 6 1 Objective 1, Stages of Meiosis Objective 1, Stages of Meiosis Prophase I: ¾ During synapsis, sometimes there is an exchange of homologous parts between non-sister chromatids. This exchange is called crossing over. 7 8 Objective 1, Stages of Meiosis Objective 1, Stages of Meiosis (2N=6) Prophase I: ¾ the spindle apparatus begins to form. ¾ the nuclear membrane breaks down: Prophase I 9 10 Objective 1, Stages of Meiosis Objective 1, 4 Possible Metaphase I Arrangements: Metaphase I: ¾ chromosomes line up along the equatorial plate in pairs, i.e.
    [Show full text]
  • The Investigational Aurora Kinase a Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant
    Published OnlineFirst February 12, 2013; DOI: 10.1158/1078-0432.CCR-12-2383 Clinical Cancer Cancer Therapy: Preclinical Research The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo Ning Zhou1, Kamini Singh2, Maria C. Mir1, Yvonne Parker5, Daniel Lindner5, Robert Dreicer5, Jeffrey A. Ecsedy6, Zhongfa Zhang7, Bin T. Teh8, Alexandru Almasan2, and Donna E. Hansel1,3,4,5 Abstract Purpose: Despite more than 70,000 new cases of bladder cancer in the United States annually, patients with advanced disease have a poor prognosis due to limited treatment modalities. We evaluated Aurora kinase A, identified as an upregulated candidate molecule in bladder cancer, as a potential therapeutic target. Experimental Design: Gene expression in human bladder cancer samples was evaluated using RNA microarray and quantitative reverse transcriptase PCR. Effects of the Aurora kinase A inhibitor MLN8237 (Millennium) on cell dynamics in malignant T24 and UM-UC-3 and papilloma-derived RT4 bladder cells were evaluated in vitro and in vivo in a mouse xenograft model. Results: A set of 13 genes involved in the mitotic spindle checkpoint, including Aurora kinases A and B, were upregulated in human urothelial carcinoma compared with normal urothelium. The Aurora kinase A inhibitor MLN8237 induced cell-cycle arrest, aneuploidy, mitotic spindle failure, and apoptosis in the human bladder cancer cell lines T24 and UM-UC-3. MLN8237 also arrested tumor growth when admin- istered orally over 4 weeks in a mouse bladder cancer xenograft model. Finally, in vitro sequential administration of MLN8237 with either paclitaxel or gemcitabine resulted in synergistic cytotoxic effects in T24 cells.
    [Show full text]
  • Lecture Disruption of Mitosis
    1 Lecture Disruption of Mitosis 1. General Information Mitosis is the process by which eukaryotic cell nuclei divide to give two daughter nuclei with identical and complete sets of chromosomes. The term mitosis, however, is commonly used to describe the entire process of cell division. This process mainly occurs in the meristematic tissues and is absolutely necessary for plant growth. Herbicides with this mode of action inhibit the formation or function of the spindle apparatus, a framework of proteins that allows segregation of the daughter chromosomes in the division process. For one herbicide with this mode of action the specific target site is unknown. 2 2. Mode of Action Microtubule Assembly Inhibitors (Interference with Spindle Fiber Formation) For herbicides with this mode of action, the prophase sequence is normal, but without the spindle apparatus, chromosomes are unable to move into the metaphase configuration and daughter chromosomes cannot migrate to their respective poles. Herbicides representing the dinitroaniline family bind to tubulin, the major microtubule protein. The herbicide-tubulin complex inhibits polymerization of microtubules at the growing end of the tubule but has no effect on depolymerization of the tubule on the other end. This leads to loss of microtubules. In contrast, the herbicides dithiopyr and pronamide do not bind to tubulin but appear to bind to another protein that may be a microtubule associated protein. The end result is shortened microtubules that cannot form the spindle fibers needed for mitosis. In both cases, after a time in the prophase state, chromosomes coalesce in the middle of the cell, cell division ceases, and growth stops.
    [Show full text]
  • Mitotic Spindles Revisited – New Insights from 3D Electron Microscopy Thomas Müller-Reichert‡, Robert Kiewisz and Stefanie Redemann*,‡
    © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383 REVIEW ARTICLE SERIES: IMAGING Mitotic spindles revisited – new insights from 3D electron microscopy Thomas Müller-Reichert‡, Robert Kiewisz and Stefanie Redemann*,‡ ABSTRACT microtubules of bipolar spindles are grouped into different classes. The mitotic spindle is a complex three-dimensional (3D) apparatus A canonical view of mitotic spindle structure in metaphase (Fig. 1) that functions to ensure the faithful segregation of chromosomes shows the following classes of microtubules (MTs): astral-MTs during cell division. Our current understanding of spindle architecture (AMTs), kinetochore-MTs (KMTs), interdigitating-MTs (IMTs) and is mainly based on a plethora of information derived from light spindle MTs (SMTs). AMTs are those microtubules that grow away microscopy with rather few insights about spindle ultrastructure from centrosomes towards the cellular cortex, thus mainly playing a obtained from electron microscopy. In this Review, we will provide role in positioning the spindle apparatus (Grill et al., 2001). The plus- insights into the history of imaging of mitotic spindles and highlight ends of KMTs are directly connected to the kinetochores (i.e. to recent technological advances in electron tomography and data specific centromeric microtubule-binding sites on the chromosomes) processing, which have delivered detailed 3D reconstructions of (Musacchio and Desai, 2017). IMTs are thought to interact with each mitotic spindles in the early embryo of the nematode Caenorhabditis other in the midzone of the spindle. This interaction is supposed to elegans. Tomographic reconstructions provide novel views on build a direct pole-to-pole connection through microtubules of spindles and will enable us to revisit and address long-standing opposite polarity (Mastronarde et al., 1993).
    [Show full text]
  • Function and Regulation of Aurora/Ipl1p Kinase Family in Cell Division
    Cell Research (2003); 13(2):69-81 http://www.cell-research.com Function and regulation of Aurora/Ipl1p kinase family in cell division 1 1 1 1,2, YU WEN KE , ZHEN DOU , JIE ZHANG , XUE BIAO YAO * 1 Laboratory for Cell Dynamics, School of Life Sciences, University of Science and Technology of China, Hefei 230027, China 2 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA ABSTRACT During mitosis, the parent cell distributes its genetic materials equally into two daughter cells through chromosome segregation, a complex movements orchestrated by mitotic kinases and its effector proteins. Faithful chromosome segregation and cytokinesis ensure that each daughter cell receives a full copy of genetic materials of parent cell. Defects in these processes can lead to aneuploidy or polyploidy. Aurora/Ipl1p family, a class of conserved serine/threonine kinases, plays key roles in chromosome segregation and cytokinesis. This article highlights the function and regulation of Aurora/Ipl1p family in mitosis and provides potential links between aberrant regulation of Aurora/Ipl1p kinases and pathogenesis of human cancer. Key words: Aurora (Ipl1p), mitosis, and cancer. INTRODUCTION among species, the regulatory machinery is conserved from yeast to human. One of most conserved regu- Cell is a fundamental unit of life that is relayed lators is serine/theronine protein kinase superfam- via mitosis. The essence of mitosis is to segregate ily that alters the function of its effectors via protein parental genomes encoded in sister chromatides into phosphorylation. Entry into mitosis is driven by pro- two daughter cells, such that each of them inherits tein kinases while initiation of exit from mitosis is one complete copy of genome.
    [Show full text]
  • LIN-5 Is a Novel Component of the Spindle Apparatus Required for Chromosome Segregation and Cleavage Plane Specification in Caenorhabditis Elegans Monique A
    LIN-5 Is a Novel Component of the Spindle Apparatus Required for Chromosome Segregation and Cleavage Plane Specification in Caenorhabditis elegans Monique A. Lorson,* H. Robert Horvitz,‡ and Sander van den Heuvel*‡ *Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129; and ‡Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Abstract. Successful divisions of eukaryotic cells re- codes a novel protein with a central coiled-coil domain. quire accurate and coordinated cycles of DNA replica- This protein localizes to the spindle apparatus in a cell tion, spindle formation, chromosome segregation, and cycle- and microtubule-dependent manner. The LIN-5 cytoplasmic cleavage. The Caenorhabditis elegans gene protein is located at the centrosomes throughout mito- lin-5 is essential for multiple aspects of cell division. sis, at the kinetochore microtubules in metaphase cells, Cells in lin-5 null mutants enter mitosis at the normal and at the spindle during meiosis. Our results show that time and form bipolar spindles, but fail chromosome LIN-5 is a novel component of the spindle apparatus alignment at the metaphase plate, sister chromatid sep- required for chromosome and spindle movements, cy- aration, and cytokinesis. Despite these defects, cells exit toplasmic cleavage, and correct alternation of the S and from mitosis without delay and progress through subse- M phases of the cell cycle. quent rounds of DNA replication, centrosome duplica- tion, and abortive mitoses. In addition, early embryos Key words: lin-5 • mitosis • cytokinesis • microtu- that lack lin-5 function show defects in spindle position- bules • mitotic checkpoint ing and cleavage plane specification.
    [Show full text]
  • Aurora B Phosphorylates Bub1 to Promote Spindle Assembly Checkpoint Signaling
    bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.425459; this version posted January 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Aurora B phosphorylates Bub1 to promote spindle assembly checkpoint signaling 2 Babhrubahan Roy, Simon J. Y. Han, Adrienne N. Fontan, Ajit P. Joglekar 3 4 Summary 5 Accurate chromosome segregation during cell division requires amphitelic attachment of each 6 chromosome to the spindle apparatus. This is ensured by the Spindle Assembly Checkpoint 7 (SAC) [1], which delays anaphase onset in response to unattached chromosomes, and an error 8 correction mechanism, which eliminates syntelic chromosome attachments [2]. The SAC is 9 activated by the Mps1 kinase. Mps1 sequentially phosphorylates the kinetochore protein 10 Spc105/KNL1 to license the recruitment of several signaling proteins including Bub1. These 11 proteins produce the Mitotic Checkpoint Complex (MCC), which delays anaphase onset [3-8]. 12 The error correction mechanism is regulated by the Aurora B kinase, which phosphorylates the 13 microtubule-binding interface of the kinetochore. Aurora B is also known to promote SAC 14 signaling indirectly [9-12]. Here we present evidence that Aurora B kinase activity directly 15 promotes MCC production in budding yeast and human cells. Using the ectopic SAC activation 16 (eSAC) system, we find that the conditional dimerization of Aurora B (or an Aurora B recruitment 17 domain) with either Bub1 or Mad1, but not the ‘MELT’ motifs in Spc105/KNL1, leads to a SAC- 18 mediated mitotic arrest [13-16].
    [Show full text]
  • The Spindle Assembly Checkpoint Review
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Current Biology 22, R966–R980, November 20, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.10.006 The Spindle Assembly Checkpoint Review Pablo Lara-Gonzalez1,2, Frederick G. Westhorpe1,2, point in the cell cycle can result in daughters with deviations and Stephen S. Taylor1,* from the normal karyotype, which in turn can result in a loss of fitness or, in the case of multicellular organisms, various diseases [2]. During mitosis and meiosis, the spindle assembly check- The solution to this challenge is sister chromatid cohesion, point acts to maintain genome stability by delaying cell mediated by cohesin, a multimeric protein ring structure that division until accurate chromosome segregation can encircles the replicated sister chromatids [3]. Following DNA be guaranteed. Accuracy requires that chromosomes replication in S phase, original and new chromatids are held become correctly attached to the microtubule spindle together by cohesin, thus maintaining their identity as apparatus via their kinetochores. When not correctly sisters. Cohesion is maintained throughout the rest of S attached to the spindle, kinetochores activate the spindle phase, G2 and into early mitosis where the chromosomes assembly checkpoint network, which in turn blocks cell align at the centre of the cell on the microtubule spindle cycle progression. Once all kinetochores become stably apparatus. At anaphase onset, the cohesin ring is opened, attached to the spindle, the checkpoint is inactivated, chromatid cohesion is lost and the sisters separate, allowing which alleviates the cell cycle block and thus allows chro- spindle forces to pull them to opposite sides of the cell (Fig- mosome segregation and cell division to proceed.
    [Show full text]