DOCSLIB.ORG

Mitotic Spindle Form and Function

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mitotic Spindle Form and Function YEASTBOOK CELL STRUCTURE & TRAFFICKING Mitotic Spindle Form and Function Mark Winey* and Kerry Bloom†,1 *Molecular Cell and Developmental Biology, University of Colorado, Boulder, Colorado 80309 and †Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280 ABSTRACT The Saccharomyces cerevisiae mitotic spindle in budding yeast is exemplified by its simplicity and elegance. Microtubules are nucleated from a crystalline array of proteins organized in the nuclear envelope, known as the spindle pole body in yeast (analogous to the centrosome in larger eukaryotes). The spindle has two classes of nuclear microtubules: kinetochore microtubules and interpolar microtubules. One kinetochore microtubule attaches to a single centromere on each chromosome, while approximately four interpolar microtubules emanate from each pole and interdigitate with interpolar microtubules from the opposite spindle to provide stability to the bipolar spindle. On the cytoplasmic face, two to three microtubules extend from the spindle pole toward the cell cortex. Processes requiring microtubule function are limited to spindles in mitosis and to spindle orientation and nuclear positioning in the cytoplasm. Microtubule function is regulated in large part via products of the 6 kinesin gene family and the 1 cytoplasmic dynein gene. A single bipolar kinesin (Cin8, class Kin-5), together with a depolymerase (Kip3, class Kin-8) or minus-end-directed kinesin (Kar3, class Kin-14), can support spindle function and cell viability. The remarkable feature of yeast cells is that they can survive with microtubules and genes for just two motor proteins, thus providing an unparalleled system to dissect microtubule and motor function within the spindle machine. TABLE OF CONTENTS Abstract 1197 Introduction 1198 Spindle Structure 1198 The Parts List 1199 Spindle pole bodies 1199 Microtubules 1203 Microtubule motor proteins 1204 Microtubule-associated proteins 1205 DNA, cohesion, and condensin springs in the spindle 1207 The kinetochore 1208 Building the Spindle 1209 Spindle pole duplication and separation 1209 Regulation of SPB duplication 1212 Spindle Dynamics 1213 Regulation of microtubule dynamics, kinetochore and interpolar microtubules 1213 Regulation of spindle length and stability 1214 Spindle Orientation and Translocation 1215 Continued Copyright © 2012 by the Genetics Society of America doi: 10.1534/genetics.111.128710 Manuscript received March 15, 2011; accepted for publication September 6, 2011 1Corresponding author: CB #3280, Coker Hall, University of North Carolina, Chapel Hill, NC 27599-3280. E-mail: [email protected] Genetics, Vol. 190, 1197–1224 April 2012 1197 CONTENTS, continued Regulation of spindle orientation and translocation 1216 Spindle Disassembly: Mitotic Exit and Preparation for the Next Cycle 1217 Prospective 1217 N the budding yeast Saccharomyces cerevisiae, the mitotic is constant for 15–20 min. At this stage the spindle appears Ispindle is exemplified by its simplicity and elegance. Mi- fairly rigid and can rotate up to 90° obliquely to the mother/ crotubules (MTs) are nucleated by the highly organized ar- bud axis. Anaphase onset is characterized by rapid linear ray of proteins inserted in a fenestra in the nuclear envelope elongation of the spindle with a velocity of 1 mm/min to known as the spindle pole body (SPB, analogous to the a length of 6 mm. These rates are consistent with earlier centrosome in larger eukaryotes). The SPB organizes two measurements taken from low-level DAPI-stained cells classes of nuclear spindle microtubules: kinetochore (KT) (Palmer et al. 1989). In anaphase the kinetochore microtu- and interpolar microtubules. One kinetochore microtubule bules shorten from their plus-end (anaphase A). As spindle attaches to a single centromere on each chromosome, while elongation ensues (anaphase B), the number of interpolar approximately four interpolar microtubules emanate from microtubules decreases to one or two interpolar microtubules each pole and interdigitate with interpolar microtubules from each pole prior to spindle disassembly. from the opposite spindle to provide stability to the bipolar The lack of detectable kinetochore structures required spindle. On the cytoplasmic face, two to three microtubules that kinetochore microtubules be detected by various compu- extend from the SPB toward the cell cortex. Processes re- tational techniques. Two key findings about the kinetochore quiring microtubule function are limited to spindles in mi- microtubules came of these analyses. First, the plus ends of tosis and spindle orientation and nuclear positioning in the the kinetochore microtubules from opposing SPBs were never cytoplasm. Microtubule function is regulated in large part found to be near each other as one might expect at metaphase via products of the 6 kinesin genes and the 1 cytoplasmic when the paired sister chromatids achieve bipolar spindle dynein gene. A single bipolar kinesin (Cin8, class Kin-5), attachment. What was observed was a gap of 250 nm be- together with a depolymerase (Kip3, class Kin-8) or mi- tween the ends of the kinetochore microtubules from each nus-end-directed kinesin (Kar3, class Kin -14), can support SPB, leading to the suggestion that the sister centromeres spindle function and cell viability. The remarkable feature were separated from each other at metaphase in yeast (Winey fi that both budding and ssion yeast cells can survive with et al. 1995). This proposal was confirmed by fluorescent mi- microtubules and genes for just a few motor proteins pro- croscopy studies of live cells with marked tubulin (Tub-GFP, vides an unparalleled system to dissect microtubule and Figure 1) centromeric DNA or kinetochores (Goshima and motor function within the spindle machine. Yanagida 2000; Tanaka et al. 2000; He et al. 2001; Pearson et al. 2001). The position of lacO arrays relative to the cen- tromere is critical to visualizing separated sister centromeres. Spindle Structure The most proximal centromere marked DNA spots [within 1 Three-dimensional ultrastructural analysis of yeast mitotic kilobase (kb) pairs from the centromere] are spatially sepa- spindle microtubules has been accomplished by reconstruc- rated for the duration of mitosis (Goshima and Yanagida tion from serial thin sections of cells (Winey et al. 1995) and 2001; Pearson et al. 2001). Second, the appearance of clusters by electron tomography of thick sections of cells (O’Toole of sister kinetochore proteins provide evidence for metaphase et al. 1999) (Figure 1). The work confirmed that kineto- in budding yeast (Pearson et al. 2001). Third, the electron chores are attached by a single microtubule as suggested tomography confirmed that anaphase A (kinetochores moving in early high-voltage electron microscopy (EM) studies of toward the poles) occurred in budding yeast (O’Toole et al. isolated yeast spindles (Peterson and Ris 1976). Reconstruc- 1999). Chromosome movement in anaphase proceeds at a rate tion of yeast mitotic spindle microtubules from serial thin of 1 mm/min. As a point of reference, if you read down this sections or electron tomograms revealed that the spindle is page at the rate a yeast chromosome moves, it would take a highly stereotypic microtubule array in these cells. In you 150 days to get to the foot of the page. Anaphase B metaphase, it is 1.4–1.5 mm long, and haploid cells contain (increasing pole-to-pole distance) is so significant in yeast 20 microtubules from each SPB. These microtubules can (the 1.5-mm metaphase spindle will lengthen to nearly be divided into shorter microtubules that do not interact 10 mm) that this movement alone may be sufficient to sepa- with antiparallel microtubules from the other SPB, suggest- rate sister chromatids. However, the reconstructions revealed ing that they are kinetochore microtubules, particularly that the kinetochore MTs of 0.5 mmlongatmetaphase since there are very nearly 16 per haploid SPB (Winey (Figure 1) are shorter in anaphase cells. In fact, they shrink et al. 1995). Once formed, the 1.5- 2.0-mm spindle length all the way to 30–50 nm in late anaphase. These remnants 1198 M. Winey and K. Bloom tween these microtubules that could be various motors or microtubule-associated proteins (MAPs), but those molecu- lar identifications have not been made (Winey et al. 1995). Finally, the long microtubules of the anaphase B central spindle seem to persist into G1 after the spindle is severed during karyokinesis. The fact that the budding yeast SPB is embedded in the nuclear envelope throughout the entire life cycle of the organism is consistent with the observation that microtubules are continually present in the nucleus. The G1 nuclear microtubule array have a median length of 150 nm (O’Toole et al. 1999). The Parts List Figure 1 Yeast mitotic spindle structure. Sixteen kinetochore microtubules and four interpolar microtubules emanate from each spindle pole in a hap- Spindle pole bodies loid cell: 40 MTs/1.5 mm spindle. (Left) The yeast mitotic spindle as seen in the electron microscope (EM) using thin sections of high-pressure frozen The S. cerevisiae SPB was first observed in the electron mi- and freeze-substituted cells (e.g.,Wineyet al. 1995). The budded cell con- croscope by Robinow and Marak (1966) (Figure 2). As the tains two SPBs (black arrows) that form a bipolar spindle (microtubules are sole microtubule-organizing center (MTOC) in these cells the filaments between the SPBs in the nucleus). The nuclear envelope (white carets) extends through the bud neck, a typical configuration at this that have a closed mitosis (the nuclear envelope stays intact point in the cell cycle. (Right) The yeast mitotic spindle as seen in the light throughout the cell cycle), the SPB must have access to the microscope using a-tubulin fused to green fluorescent protein (Tub-GFP, nucleoplasm to form the microtubules of the mitotic spindle shown in Figure as white protein on black background). The outline of the and to the cytoplasm to form the astral microtubules that cell is illustrated by the outline in white. The spindle can be seen at the bud will position the nucleus.
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]
  • Effects of Activation of the LINE-1 Antisense Promoter on the Growth of Cultured Cells
    www.nature.com/scientificreports OPEN Efects of activation of the LINE‑1 antisense promoter on the growth of cultured cells Tomoyuki Honda1*, Yuki Nishikawa1, Kensuke Nishimura1, Da Teng1, Keiko Takemoto2 & Keiji Ueda1 Long interspersed element 1 (LINE‑1, or L1) is a retrotransposon that constitutes ~ 17% of the human genome. Although ~ 6000 full‑length L1s spread throughout the human genome, their biological signifcance remains undetermined. The L1 5′ untranslated region has bidirectional promoter activity with a sense promoter driving L1 mRNA production and an antisense promoter (ASP) driving the production of L1‑gene chimeric RNAs. Here, we stimulated L1 ASP activity using CRISPR‑Cas9 technology to evaluate its biological impacts. Activation of the L1 ASP upregulated the expression of L1 ASP‑driven ORF0 and enhanced cell growth. Furthermore, the exogenous expression of ORF0 also enhanced cell growth. These results indicate that activation of L1 ASP activity fuels cell growth at least through ORF0 expression. To our knowledge, this is the frst report demonstrating the role of the L1 ASP in a biological context. Considering that L1 sequences are desilenced in various tumor cells, our results indicate that activation of the L1 ASP may be a cause of tumor growth; therefore, interfering with L1 ASP activity may be a potential strategy to suppress the growth. Te human genome contains many transposable element-derived sequences, such as endogenous retroviruses and long interspersed element 1 (LINE-1, or L1). L1 is one of the major classes of retrotransposons, and it constitutes ~ 17% of the human genome1. Full-length L1 consists of a 5′ untranslated region (UTR), two open reading frames (ORFs) that encode the proteins ORF1p and ORF2p, and a 3′ UTR with a polyadenylation signal.
    [Show full text]
  • Integrating Single-Step GWAS and Bipartite Networks Reconstruction Provides Novel Insights Into Yearling Weight and Carcass Traits in Hanwoo Beef Cattle
    animals Article Integrating Single-Step GWAS and Bipartite Networks Reconstruction Provides Novel Insights into Yearling Weight and Carcass Traits in Hanwoo Beef Cattle Masoumeh Naserkheil 1 , Abolfazl Bahrami 1 , Deukhwan Lee 2,* and Hossein Mehrban 3 1 Department of Animal Science, University College of Agriculture and Natural Resources, University of Tehran, Karaj 77871-31587, Iran; [email protected] (M.N.); [email protected] (A.B.) 2 Department of Animal Life and Environment Sciences, Hankyong National University, Jungang-ro 327, Anseong-si, Gyeonggi-do 17579, Korea 3 Department of Animal Science, Shahrekord University, Shahrekord 88186-34141, Iran; [email protected] * Correspondence: [email protected]; Tel.: +82-31-670-5091 Received: 25 August 2020; Accepted: 6 October 2020; Published: 9 October 2020 Simple Summary: Hanwoo is an indigenous cattle breed in Korea and popular for meat production owing to its rapid growth and high-quality meat. Its yearling weight and carcass traits (backfat thickness, carcass weight, eye muscle area, and marbling score) are economically important for the selection of young and proven bulls. In recent decades, the advent of high throughput genotyping technologies has made it possible to perform genome-wide association studies (GWAS) for the detection of genomic regions associated with traits of economic interest in different species. In this study, we conducted a weighted single-step genome-wide association study which combines all genotypes, phenotypes and pedigree data in one step (ssGBLUP). It allows for the use of all SNPs simultaneously along with all phenotypes from genotyped and ungenotyped animals. Our results revealed 33 relevant genomic regions related to the traits of interest.
    [Show full text]
  • NIH Public Access Author Manuscript Structure
    NIH Public Access Author Manuscript Structure. Author manuscript; available in PMC 2014 June 04. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Structure. 2013 June 4; 21(6): 939–950. doi:10.1016/j.str.2013.04.018. A Cryptic TOG Domain with a Distinct Architecture Underlies CLASP- Dependent Bipolar Spindle Formation Jonathan B. Leano1, Stephen L. Rogers2,3,4, and Kevin C. Slep2 1Dept. of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599 2Dept. of Biology, University of North Carolina, Chapel Hill, NC 27599 3Carolina Center for Genome Science, University of North Carolina, Chapel Hill, NC 27599 4Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599 SUMMARY CLASP is a key regulator of microtubule (MT) dynamics and bipolar mitotic spindle structure with CLASP mutants displaying a distinctive monopolar spindle phenotype. It has been postulated that cryptic TOG domains underlie CLASP’s ability regulate MT dynamics. Here, we report the crystal structure of the first cryptic TOG domain (TOG2) from human CLASP1, revealing the existence of a bona fide TOG array in the CLASP family. Strikingly, CLASP1 TOG2 exhibits a unique, convex architecture across the tubulin-binding surface that contrasts with the flat tubulin- binding surface of XMAP215 family TOG domains. Mutations in key, conserved TOG2 determinants abrogate the ability of CLASP mutants to rescue bipolar spindle formation in Drosophila cells depleted of endogenous CLASP. These findings highlight the common mechanistic use of TOG domains in XMAP215 and CLASP families to regulate MT dynamics, and suggest that differential TOG domain architecture may confer distinct functions to these critical cytoskeletal regulators.
    [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]
  • CLASP Promotes Microtubule Bundling in Metaphase Spindle Independently of Ase1/PRC1 in Fission Yeast Hirohisa Ebina1, Liang Ji1,2 and Masamitsu Sato1,2,3,4,*
    © 2019. Published by The Company of Biologists Ltd | Biology Open (2019) 8, bio045716. doi:10.1242/bio.045716 RESEARCH ARTICLE CLASP promotes microtubule bundling in metaphase spindle independently of Ase1/PRC1 in fission yeast Hirohisa Ebina1, Liang Ji1,2 and Masamitsu Sato1,2,3,4,* ABSTRACT centrosomes or spindle pole bodies (SPBs). Spindle MTs are then Microtubules in the mitotic spindle are organised by microtubule- augmented by a number of MAPs promoting MT nucleation, associated proteins. In the late stage of mitosis, spindle microtubules controlling the dynamic property of MTs, and bundling MT are robustly organised through bundling by the antiparallel filaments. All of those processes are required to establish bipolarity microtubule bundler Ase1/PRC1. In early mitosis, however, it is not and the robust spindle structure, as failures in either process may well characterised as to whether spindle microtubules are actively result in unequal segregation of chromosomes (McDonald and bundled, as Ase1 does not particularly localise to the spindle at that McIntosh, 1993; McDonald et al., 1992). stage. Here we show that the conserved microtubule-associated MT bundling is particularly essential to stabilise and maintain the protein CLASP (fission yeast Peg1/Cls1) facilitates bundling of structural backbone of the spindle. MTs emanating from each spindle microtubules in early mitosis. The peg1 mutant displayed a spindle pole are connected through bundling mostly at the centre of fragile spindle with unbundled microtubules, which eventually the spindle, throughout mitosis. Several proteins promoting MT resulted in collapse of the metaphase spindle and abnormal bundling have been identified to date. segregation of chromosomes. Peg1 is known to be recruited to the The conserved non-motor MAP Ase1/PRC1 preferentially spindle by Ase1 to stabilise antiparallel microtubules in late mitosis.
    [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]
  • Noncoding Rnas As Novel Pancreatic Cancer Targets
    NONCODING RNAS AS NOVEL PANCREATIC CANCER TARGETS by Amy Makler A Thesis Submitted to the Faculty of The Charles E. Schmidt College of Science In Partial Fulfillment of the Requirements for the Degree of Master of Science Florida Atlantic University Boca Raton, FL August 2018 Copyright 2018 by Amy Makler ii ACKNOWLEDGEMENTS I would first like to thank Dr. Narayanan for his continuous support, constant encouragement, and his gentle, but sometimes critical, guidance throughout the past two years of my master’s education. His faith in my abilities and his belief in my future success ensured I continue down this path of research. Working in Dr. Narayanan’s lab has truly been an unforgettable experience as well as a critical step in my future endeavors. I would also like to extend my gratitude to my committee members, Dr. Binninger and Dr. Jia, for their support and suggestions regarding my thesis. Their recommendations added a fresh perspective that enriched our initial hypothesis. They have been indispensable as members of my committee, and I thank them for their contributions. My parents have been integral to my successes in life and their support throughout my education has been crucial. They taught me to push through difficulties and encouraged me to pursue my interests. Thank you, mom and dad! I would like to thank my boyfriend, Joshua Disatham, for his assistance in ensuring my writing maintained a logical progression and flow as well as his unwavering support. He was my rock when the stress grew unbearable and his encouraging words kept me pushing along.
    [Show full text]
  • A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family
    Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2018 A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family Linda Molla Follow this and additional works at: https://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Linda Molla June 2018 © Copyright by Linda Molla 2018 A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY Linda Molla, Ph.D. The Rockefeller University 2018 APOBEC2 is a member of the AID/APOBEC cytidine deaminase family of proteins. Unlike most of AID/APOBEC, however, APOBEC2’s function remains elusive. Previous research has implicated APOBEC2 in diverse organisms and cellular processes such as muscle biology (in Mus musculus), regeneration (in Danio rerio), and development (in Xenopus laevis). APOBEC2 has also been implicated in cancer. However the enzymatic activity, substrate or physiological target(s) of APOBEC2 are unknown. For this thesis, I have combined Next Generation Sequencing (NGS) techniques with state-of-the-art molecular biology to determine the physiological targets of APOBEC2. Using a cell culture muscle differentiation system, and RNA sequencing (RNA-Seq) by polyA capture, I demonstrated that unlike the AID/APOBEC family member APOBEC1, APOBEC2 is not an RNA editor. Using the same system combined with enhanced Reduced Representation Bisulfite Sequencing (eRRBS) analyses I showed that, unlike the AID/APOBEC family member AID, APOBEC2 does not act as a 5-methyl-C deaminase.
    [Show full text]