DOCSLIB.ORG

Microtubule Organization and the Distribution of Γ-Tubulin in Spermatogenesis of a Beetle, Tenebrio Molitor (Tenebrionidae, Coleoptera, Insecta)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Microtubule Organization and the Distribution of Γ-Tubulin in Spermatogenesis of a Beetle, Tenebrio Molitor (Tenebrionidae, Coleoptera, Insecta) Journal of Cell Science 108, 3855-3865 (1995) 3855 Printed in Great Britain © The Company of Biologists Limited 1995 JCS3135 Microtubule organization and the distribution of γ-tubulin in spermatogenesis of a beetle, Tenebrio molitor (Tenebrionidae, Coleoptera, Insecta) Klaus Werner Wolf1,* and Harish C. Joshi2 1Institut für Biologie, Medizinische Universität Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Deutschland 2Department of Anatomy and Cell Biology, Emory University, School of Medicine, Atlanta, Georgia 30322, USA *Author for correspondence SUMMARY The present study focuses on the restructuring of the prominent cytoplasmic MT system of primary spermato- microtubule (MT) cytoskeleton and microtubule-organiz- cytes in prophase, microtubule nucleation apparently ing centres (MTOCs) throughout spermatogenesis of a occurs in the absence of immunologically detectable γ- darkling beetle, Tenebrio molitor (Tenebrionidae, tubulin. At the poles of the meiotic spindles, MTs are Coleoptera, Insecta). To this end, serial ultrathin sections directly inserted into γ-tubulin-containing material and this through male germ cells were studied using transmission connection is considered responsible for their nucleation. electron microscopy. Additionaly, spindles and young sper- The interzone spindle MTs of telophase cells contain γ- matids were isolated from testes under MT-stabilizing con- tubulin and this may confer stability to them. Finally, ditions and doubly labeled with antibodies against β- and manchette MTs of spermatids originate in the vicinity of γ-tubulin. The latter is a tubulin isoform detected in the acrosome precursor but are not inserted into this body. MTOCs of a wide variety of species. The observations The acrosome precursor is surrounded by a membrane and suggest that microtubules may be nucleated from sites with is clearly detected by the antibody against γ-tubulin. and without high γ-tubulin content and that these sites do not necessarily possess canonical centrosomes. In a Key words: acrosome, centrosome, meiosis, spermatid INTRODUCTION spindles which bring about chromosome segregation during two sucessive divisions. The cells arising from the second All eukaryotic cells possess skeletal elements in the form of meiotic division develop into spermatids. These are slender microtubles (MTs). Most MTs have one end close to an element elongated cells containing a haploid genome. Spermatids which is believed to organize the microtubular cytoskeleton. finally transform into spermatozoa ready for fertilization. The The expressions centrosome (e.g. Mazia, 1984) or microtubule- basic cytological features of canonical spermatogenesis are organizing-centre (MTOCs) (Pickett-Heaps, 1969) are widely well known. The present study is aimed at the restructuring of used in this context. The last few years have seen the discovery the microtubular cytoskeleton during spermatogenesis of the of a series of components contained within MTOCs (for a darkling beetle Tenebrio molitor (Tenebrionidae, Coleoptera, review, see Kalt and Schliwa, 1993). A highly conserved Insecta). This organism has been selected on technical tubulin isoform, γ-tubulin, has been found to be enriched within grounds: rearing is easy and its size renders it a handy system MTOCs from a wide variety of species and cell types (for a for cytological work. In addition, conventional bipolar spindles review, see Joshi, 1994). The consistent presence of γ-tubulin are formed in male meiosis of the beetle (Wolf and Hellwage, in MTOCs renders it a prime candidate for a role in MT 1995) and sperm structure is regular (Baccetti et al., 1973). nucleation. In order to gain further insight into the function of Thus, T. molitor has the potential of serving as a model γ-tubulin, it appears worthwhile to study its distribution in organism for Coleoptera, the largest insect order with more relation to the changes in the microtubular cytoskeleton in a than 350,000 species described so far. A fine structure analysis complex developmental process such as spermatogenesis. A of MTOCs in spermatogenesis of T. molitor supplements the polyclonal antibody, generated against a conserved portion of immunological work. γ-tubulin (Joshi et al., 1992), renders this feasible. The antibody used in this study was made against a peptide that was carefully chosen to be unique to γ-tubulin (Table 1). MATERIALS AND METHODS During spermatogenesis, profound alterations of the cells in terms of size, shape and function occur. Primary spermatocytes The experimental animal in prophase are characterized by the pairing of homologous Male pupae from a laboratory strain of Tenebrio molitor, reared on chromosomes. Cells of the ensuing stages possess meiotic rolled oats, were used in this study. 3856 K. W. Wolf and H. C. Joshi Electron microscopy Table 1. Peptide used to raise the antibody to γ- Pupal testes were prepared for electron microscopy as reported pre- tubulin and C-terminal peptides of γ-tubulins from yeast viously for Lepidopteran tissue (Wolf, 1994). to man Anti-tubulin immunofluorescence Peptide EEFATEGTDRKDVFFY-C Human EEFATEGTDRKDVFFY The testes were dissected out in Ringer solution (Wolf, 1994), trans- Mouse EEFATEGTDRKDVFFY ferred into a microtubule-stabilizing buffer (100 mM piperazine-N,N′ Drosophila EDFANDGLDRKDVFFY bis(2-ethane sulfonic acid), pH 6.8, 1 mM MgSO4, 1 mM ethylene Aspergillus EEFATEGGDRKDVFFY glycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid, 1% Triton X- Yeast (S. pombe) ESFATEGVDRKDVFFY 100) and minced using tungsten needles. The suspension containing α-tubulin SDKTIGGDDSFNTFF germ cells was spun onto coverslips in a cytocentrifuge (Shandon D L D β GTSDAQLERISVXYNE Cytospin II, 1,500 rpm, 5 minutes). Fixation was carried out with -tubulin 0.25% glutaraldehyde and 2% formaldehyde in microtubule-stabiliz- The antibody to γ-tubulin used in this study was elicited from a C-terminal ing buffer devoid of detergent (30 minutes). For further stabilization peptide conserved among γ-tubulins from yeast to man. The comparison with of the cytoskeletons, the coverslips were subsequently immersed in peptides from α- and β-tubulin shows that cross-reactions are not likely. 100% methanol (−20°C). In order to block free aldehyde residues, the From Joshi et al. (1992). cells were treated with NaBH4 (0.5 mg/ml in phosphate-buffered saline (PBS) according to the method of Osborn and Weber (1982). Following this and all subsequent incubations, the coverslips were Prometaphase primary spermatocytes are characterized by rinsed three times (5 minutes) in PBS containing 0.02% NaN3 and the presence of an elongated microtubular spindle and chro- 0.1% Triton X-100 and once (5 minutes) in this solution without mosomes scattered throughout the spindle area (Fig. 1d-f). The detergent. remainder of the cytoplasm is virtually devoid of MTs. The Double labeling of γ- and β-tubulin was achieved by using simul- spindle poles are intensely labeled with the antibody against γ- taneously two different antibodies. These were: (1) a mouse mono- tubulin (Fig. 1e). This is also true for metaphase in primary clonal antibody against β-tubulin (Sigma); and (2) a rabbit polyclonal γ spermatocytes, when a regular bipolar spindle forms and the antibody against -tubulin (Joshi et al., 1992). The antibodies were bivalents are aligned in the equatorial plate (Fig. 1g-i). Spindle diluted 1:100 with PBS containing 10 mg/ml bovine serum albumine (BSA) prior to use. structure does not change much in early anaphase, but half Incubation with a biotin-conjugated anti-mouse antibody (Sigma) at bivalents separate from one another (Fig. 1j-l). In primary sper- a dilution of 1:50 in PBS containing 10 mg/ml BSA (1 hour) and finally matocytes in mid telophase I, the spindle elongates and the with rhodamine-coupled avidine (Sigma) diluted 1:50 with PBS con- chromosomes reach the spindle poles. A prominent interzone taining 10 mg/ml BSA (1 hour) was used to visualize the binding of spindle develops between the prospective daughter nuclei. The the antibody against β-tubulin. The specimens were incubated with 1 antibody against γ-tubulin detects two separate fluorescent spots mg/ml polylysine (Serva) in PBS (10 minutes) prior to the last step. The per spindle pole. Additionally, there is label at the poleward antibody against γ-tubulin was rendered visible through a fluorescein- ends of the interzone spindle at the equatorial face of the isothiocyanate (FITC)-conjugated anti-rabbit antibody. In order to µ ′ prospective daughter nuclei (Fig. 1m-o). Primary spermatocytes visualize the chromatin, the specimens were stained with 5 g/ml 4 ,6- in late telophase show two weak fluorescent spots close to the diamidino-2-phenylindole.2 HCl (DAPI) (Serva) in citrate buffer (100 daughter nuclei when labeled with the antibody against γ- mM citric acid, 200 mM Na2HPO4, pH7). The specimens were mounted in PBS AF3 (Citifluor Ltd, London). The preparations were sealed with tubulin. The two spots are further apart from one another than nail varnish. Except for the methanol treatment, all reactions were in the previous stage. The interzone spindle fluoresces faintly carried out at room temperature. The cells were analysed and pho- throughout (Fig. 1p-r). The comparison of spindles in the first tographed using a Laborlux 12 photomicroscope equipped with epiflu- meiosis (Fig. 1a-c to p-r) with spindles stained under identical orescence illumination and a Fluotar ×100 (Leitz, Wetzlar). conditions but with the antibody to γ-tubulin omitted (see Fig. 8a-c) showed that the weak
Load more

Recommended publications
  • Expression and Localization of Myosin VI in Developing Mouse Spermatids
    Histochem Cell Biol DOI 10.1007/s00418-017-1579-z ORIGINAL PAPER Expression and localization of myosin VI in developing mouse spermatids Przemysław Zakrzewski1 · Robert Lenartowski2 · Maria Jolanta Re˛dowicz3 · Kathryn G. Miller4 · Marta Lenartowska1 Accepted: 4 May 2017 © The Author(s) 2017. This article is an open access publication Abstract Myosin VI (MVI) is a versatile actin-based and Sertoli cell actin hoops. Since this is the frst report of motor protein that has been implicated in a variety of dif- MVI expression and localization during mouse spermio- ferent cellular processes, including endo- and exocytic genesis and MVI partners in developing sperm have not yet vesicle traffcking, Golgi morphology, and actin structure been identifed, we discuss some probable roles for MVI stabilization. A role for MVI in crucial actin-based pro- in this process. During early stages, MVI is hypothesized cesses involved in sperm maturation was demonstrated in to play a role in Golgi morphology and function as well Drosophila. Because of the prominence and importance of as in actin dynamics regulation important for attachment actin structures in mammalian spermiogenesis, we inves- of developing acrosome to the nuclear envelope. Next, tigated whether MVI was associated with actin-mediated the protein might also play anchoring roles to help gener- maturation events in mammals. Both immunofuorescence ate forces needed for spermatid head elongation. Moreo- and ultrastructural analyses using immunogold labeling ver, association of MVI with actin that accumulates in showed that MVI was strongly linked with key structures the Sertoli cell ectoplasmic specialization and other actin involved in sperm development and maturation.
    [Show full text]
  • “Salivary Gland Cellular Architecture in the Asian Malaria Vector Mosquito Anopheles Stephensi”
    Wells and Andrew Parasites & Vectors (2015) 8:617 DOI 10.1186/s13071-015-1229-z RESEARCH Open Access “Salivary gland cellular architecture in the Asian malaria vector mosquito Anopheles stephensi” Michael B. Wells and Deborah J. Andrew* Abstract Background: Anopheles mosquitoes are vectors for malaria, a disease with continued grave outcomes for human health. Transmission of malaria from mosquitoes to humans occurs by parasite passage through the salivary glands (SGs). Previous studies of mosquito SG architecture have been limited in scope and detail. Methods: We developed a simple, optimized protocol for fluorescence staining using dyes and/or antibodies to interrogate cellular architecture in Anopheles stephensi adult SGs. We used common biological dyes, antibodies to well-conserved structural and organellar markers, and antibodies against Anopheles salivary proteins to visualize many individual SGs at high resolution by confocal microscopy. Results: These analyses confirmed morphological features previously described using electron microscopy and uncovered a high degree of individual variation in SG structure. Our studies provide evidence for two alternative models for the origin of the salivary duct, the structure facilitating parasite transport out of SGs. We compare SG cellular architecture in An. stephensi and Drosophila melanogaster, a fellow Dipteran whose adult SGs are nearly completely unstudied, and find many conserved features despite divergence in overall form and function. Anopheles salivary proteins previously observed at the basement membrane were localized either in SG cells, secretory cavities, or the SG lumen. Our studies also revealed a population of cells with characteristics consistent with regenerative cells, similar to muscle satellite cells or midgut regenerative cells. Conclusions: This work serves as a foundation for linking Anopheles stephensi SG cellular architecture to function and as a basis for generating and evaluating tools aimed at preventing malaria transmission at the level of mosquito SGs.
    [Show full text]
  • The Intestinal Protozoa
    The Intestinal Protozoa A. Introduction 1. The Phylum Protozoa is classified into four major subdivisions according to the methods of locomotion and reproduction. a. The amoebae (Superclass Sarcodina, Class Rhizopodea move by means of pseudopodia and reproduce exclusively by asexual binary division. b. The flagellates (Superclass Mastigophora, Class Zoomasitgophorea) typically move by long, whiplike flagella and reproduce by binary fission. c. The ciliates (Subphylum Ciliophora, Class Ciliata) are propelled by rows of cilia that beat with a synchronized wavelike motion. d. The sporozoans (Subphylum Sporozoa) lack specialized organelles of motility but have a unique type of life cycle, alternating between sexual and asexual reproductive cycles (alternation of generations). e. Number of species - there are about 45,000 protozoan species; around 8000 are parasitic, and around 25 species are important to humans. 2. Diagnosis - must learn to differentiate between the harmless and the medically important. This is most often based upon the morphology of respective organisms. 3. Transmission - mostly person-to-person, via fecal-oral route; fecally contaminated food or water important (organisms remain viable for around 30 days in cool moist environment with few bacteria; other means of transmission include sexual, insects, animals (zoonoses). B. Structures 1. trophozoite - the motile vegetative stage; multiplies via binary fission; colonizes host. 2. cyst - the inactive, non-motile, infective stage; survives the environment due to the presence of a cyst wall. 3. nuclear structure - important in the identification of organisms and species differentiation. 4. diagnostic features a. size - helpful in identifying organisms; must have calibrated objectives on the microscope in order to measure accurately.
    [Show full text]
  • Oecd Guideline for the Testing of Chemicals
    474 Adopted: 21st July 1997 OECD GUIDELINE FOR THE TESTING OF CHEMICALS Mammalian Erythrocyte Micronucleus Test INTRODUCTION 1. The mammalian in vivo micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents. 2. The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. 3. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. 4. Definitions used are set out in the Annex. INITIAL CONSIDERATIONS 5. The bone marrow of rodents is routinely used in this test since polychromatic erythrocytes are produced in that tissue. The measurement of micronucleated immature (polychromatic) erythrocytes in peripheral blood is equally acceptable in any species in which the inability of the spleen to remove micronucleated erythrocytes has been demonstrated, or which has shown an adequate sensitivity to detect agents that cause structural or numerical chromosome aberrations. Micronuclei can be distinguished by a number of criteria. These include identification of the presence or absence of a kinetochore or centromeric DNA in the micronuclei. The frequency of micronucleated immature (polychromatic) erythrocytes is the principal endpoint.
    [Show full text]
  • S2(R1) Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
    Guidance for Industry S2(R1) Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) June 2012 ICH Guidance for Industry S2(R1) Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use Additional copies are available from: Office of Communications Division of Drug Information, WO51, Room 2201 Center for Drug Evaluation and Research Food and Drug Administration 10903 New Hampshire Ave., Silver Spring, MD 20993-0002 Phone: 301-796-3400; Fax: 301-847-8714 [email protected] http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm and/or Office of Communication, Outreach and Development, HFM-40 Center for Biologics Evaluation and Research Food and Drug Administration 1401 Rockville Pike, Rockville, MD 20852-1448 http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm (Tel) 800-835-4709 or 301-827-1800 U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) June 2012 ICH Contains Nonbinding Recommendations TABLE OF CONTENTS I. INTRODUCTION (1)....................................................................................................... 1 A. Objectives of the Guidance (1.1)...................................................................................................1
    [Show full text]
  • Biology Chapter 19 Kingdom Protista Domain Eukarya Description Kingdom Protista Is the Most Diverse of All the Kingdoms
    Biology Chapter 19 Kingdom Protista Domain Eukarya Description Kingdom Protista is the most diverse of all the kingdoms. Protists are eukaryotes that are not animals, plants, or fungi. Some unicellular, some multicellular. Some autotrophs, some heterotrophs. Some with cell walls, some without. Didinium protist devouring a Paramecium protist that is longer than it is! Read about it on p. 573! Where Do They Live? • Because of their diversity, we find protists in almost every habitat where there is water or at least moisture! Common Examples • Ameba • Algae • Paramecia • Water molds • Slime molds • Kelp (Sea weed) Classified By: (DON’T WRITE THIS DOWN YET!!! • Mode of nutrition • Cell walls present or not • Unicellular or multicellular Protists can be placed in 3 groups: animal-like, plantlike, or funguslike. Didinium, is a specialist, only feeding on Paramecia. They roll into a ball and form cysts when there is are no Paramecia to eat. Paramecia, on the other hand are generalists in their feeding habits. Mode of Nutrition Depends on type of protist (see Groups) Main Groups How they Help man How they Hurt man Ecosystem Roles KEY CONCEPT Animal-like protists = PROTOZOA, are single- celled heterotrophs that can move. Oxytricha Reproduce How? • Animal like • Unicellular – by asexual reproduction – Paramecium – does conjugation to exchange genetic material Animal-like protists Classified by how they move. macronucleus contractile vacuole food vacuole oral groove micronucleus cilia • Protozoa with flagella are zooflagellates. – flagella help zooflagellates swim – more than 2000 zooflagellates • Some protists move with pseudopods = “false feet”. – change shape as they move –Ex. amoebas • Some protists move with pseudopods.
    [Show full text]
  • I Vigour. by This Means We Provide Not Only a Break Which Membrane Could Be Made Out
    16 DR. G. C. CHATTERJEE: THE CULTIVATION OF TRYPANOSOMA, ETC. lilled with bluish-stained granules. No other structure could THE CULTIVATION OF TRYPANOSOMA be made out. The posterior end (that opposite to the OUT OF THE LEISHMAN-DONOVAN aagellum end) was finely drawn out as it were into a small flagellum. BODY UPON THE METHOD OF In the moist hanging-drop preparation made on the third CAPTAIN L. ROGERS, I.M.S. lay the appearance of the fully developed parasite was most interesting. In examining the specimen under 1/16 th inch BY G. C. CHATTERJEE, M.B., oil immersion lens I found several elongated flagellate ASSISTANT BACTERIOLOGIST, MEDICAL COLLEGE, CALCUTTA. bodies moving slowly across the field by a lashing side-to- side movement of the this end the front (With Coloured Illustration.) flagellum, being of the moving parasite. The movement was distinctly slow, much slower than that of an FOLLOWING the method of L. ordinary’ trypanosoma my teacher, Captain Rogers, (trvpanosoma Brucei or Evansi). No wriggling movement I.M.S., for cultivating Leishman-Donovan bodies in citrate could be made out. The thick flagellum end was ’distinctly of sodium solution I have succeeded in developing trypano-. seen moving among the broken-down red corpuscles which soma from the Leishman-Donovan bodies. were pushed out by the jerking movement of the flagellum. In one field .I a a The patient from whom the blood for making the culture found group of these parasites lying in clump, their anterior ends being free, reminding one was taken by spleen puncture was an inhabitant of Sylhet.
    [Show full text]
  • Development of a Novel Micronucleus Assay in the Human 3-D Skin Model, Epidermtm
    Development of a Novel Micronucleus Assay in the Human 3-D Skin Model, EpiDermTM. R D Curren1, G Mun1, D P Gibson2, and M J Aardema2. 1Institute for In VitroSciences, Inc., Gaithersburg, MD; 2The Procter & Gamble Co., Cincinnati, OH. Presented at the 44nd Annual Meeting of the Society of Toxicology New Orleans, Louisiana March 6-10, 2005 Abstract The rodent in vivo micronucleus assay is an important part of a tiered testing strategy in genetic toxicology. However, this assay, in general, only provides information about materials available systemically, not at the point of contact, e.g. skin. Although in vivo rodent skin micronucleus assays are being developed, the results will still require extrapolation to the human. Furthermore, to fully comply with recent European legislation such as the 7th Amendment to the Cosmetics Directive, non-animal test methods will be needed to assess new chemicals and ingredients. Therefore we have begun development of a micronucleus assay using a commercially available 3-D engineered skin model of human origin, EpiDermTM (MatTek Corp, Ashland, MA). We first evaluated whether a population of binucleated cells sufficient for a micronucleus assay could be obtained by exposing the tissue to 1-3 ug/ml cytochalasin B (Cyt B). The frequency of binucleated cells increased both with time (to at least 120 h) and with increasing concentration of Cyt B. Three ug/ml Cyt B allowed us to reliably obtain 40-50% binucleated cells at 48h. Mitomycin C (MMC) was then used (in the presence of 3 ug/ml Cyt B) to investigate toxicity and micronuclei formation in EpiDermTM.
    [Show full text]
  • Ultrastrucure of Germ Cells During Spermatogenesis
    Korean J. Malacol. 26(1): 33-43, 2010 Ultrastrucure of Germ Cells during Spermatogenesis and Some Characteristics of Sperm Morphology in Male Mytilus coruscus (Bivalvia: Mytilidae) on the West Coast of Korea Jin Hee Kim1, Ee-Yung Chung2, Ki-Ho Choi3, Kwan Ha Park4, and Sung-Woo Park4 1Korea Inter-University Unstitute of Ocean Science, Pukyong National University, Busan 608-737, Korea 2Korea Marine Environment & Ecosystem Institute, Dive Korea, Bucheon 420-857, Korea 3West Sea Fisheries Research Institute, National Fisheries Research and Development Institute, Incheon 400-420, Korea 4Department of Aquatic Life Medicine, Kunsan National University, Kunsan 573-701, Korea ABSTRACT The ultrastructure of germ cells during spermatogenesis and some characteristics of sperm morphology in male Mytilus coruscus, which was collected on the coastal waters of Gyeokpo in western Korea, were investigated by transmission electron microscope observations. The morphology of the spermatozoon has a primitive type and is similar to those of other bivalves in that it contains a short midpiece with five mitochondria surrounding the centrioles. The morphologies of the sperm nucleus type and the acrosome shape of this species have an oval and modified cone shape, respectively. In particular, the axial rod is observed between the nucleus and acrosome of the sperm. The spermatozoon is approximately 45-50 μm in length including a sperm nucleus (about 1.46 μm in length), an acrosome (about 3.94 μm in length) and tail flagellum (approximately 40-45 μm). The axoneme of the sperm tail flagellum consists of nine pairs of microtubules at the periphery and a pair at the center.
    [Show full text]
  • Nuclear Isoforms of Neurofibromin Are Required for Proper Spindle Organization and Chromosome Segregation
    cells Article Nuclear Isoforms of Neurofibromin Are Required for Proper Spindle Organization and Chromosome Segregation Charoula Peta, Emmanouella Tsirimonaki, Dimitris Samouil, Kyriaki Georgiadou and Dimitra Mangoura * Basic Research Center, Biomedical Research Foundation of the Academy of Athens, 4 Soranou Ephessiou, 11527 Athens, Greece; [email protected] (C.P.); [email protected] (E.T.); [email protected] (D.S.); [email protected] (K.G.) * Correspondence: [email protected]; Tel.: +30-210-659-7087 Received: 18 July 2020; Accepted: 22 October 2020; Published: 23 October 2020 Abstract: Mitotic spindles are highly organized, microtubule (MT)-based, transient structures that serve the fundamental function of unerring chromosome segregation during cell division and thus of genomic stability during tissue morphogenesis and homeostasis. Hence, a multitude of MT-associated proteins (MAPs) regulates the dynamic assembly of MTs in preparation for mitosis. Some tumor suppressors, normally functioning to prevent tumor development, have now emerged as significant MAPs. Among those, neurofibromin, the product of the Neurofibromatosis-1 gene (NF1), a major Ras GTPase activating protein (RasGAP) in neural cells, controls also the critical function of chromosome congression in astrocytic cellular contexts. Cell type- and development-regulated splicings may lead to the inclusion or exclusion of NF1exon51, which bears a nuclear localization sequence (NLS) for nuclear import at G2; yet the functions of the produced NLS and DNLS neurofibromin isoforms have not been previously addressed. By using a lentiviral shRNA system, we have generated glioblastoma SF268 cell lines with conditional knockdown of NLS or DNLS transcripts. In dissecting the roles of NLS or DNLS neurofibromins, we found that NLS-neurofibromin knockdown led to increased density of cytosolic MTs but loss of MT intersections, anastral spindles featuring large hollows and abnormal chromosome positioning, and finally abnormal chromosome segregation and increased micronuclei frequency.
    [Show full text]
  • Nomina Histologica Veterinaria, First Edition
    NOMINA HISTOLOGICA VETERINARIA Submitted by the International Committee on Veterinary Histological Nomenclature (ICVHN) to the World Association of Veterinary Anatomists Published on the website of the World Association of Veterinary Anatomists www.wava-amav.org 2017 CONTENTS Introduction i Principles of term construction in N.H.V. iii Cytologia – Cytology 1 Textus epithelialis – Epithelial tissue 10 Textus connectivus – Connective tissue 13 Sanguis et Lympha – Blood and Lymph 17 Textus muscularis – Muscle tissue 19 Textus nervosus – Nerve tissue 20 Splanchnologia – Viscera 23 Systema digestorium – Digestive system 24 Systema respiratorium – Respiratory system 32 Systema urinarium – Urinary system 35 Organa genitalia masculina – Male genital system 38 Organa genitalia feminina – Female genital system 42 Systema endocrinum – Endocrine system 45 Systema cardiovasculare et lymphaticum [Angiologia] – Cardiovascular and lymphatic system 47 Systema nervosum – Nervous system 52 Receptores sensorii et Organa sensuum – Sensory receptors and Sense organs 58 Integumentum – Integument 64 INTRODUCTION The preparations leading to the publication of the present first edition of the Nomina Histologica Veterinaria has a long history spanning more than 50 years. Under the auspices of the World Association of Veterinary Anatomists (W.A.V.A.), the International Committee on Veterinary Anatomical Nomenclature (I.C.V.A.N.) appointed in Giessen, 1965, a Subcommittee on Histology and Embryology which started a working relation with the Subcommittee on Histology of the former International Anatomical Nomenclature Committee. In Mexico City, 1971, this Subcommittee presented a document entitled Nomina Histologica Veterinaria: A Working Draft as a basis for the continued work of the newly-appointed Subcommittee on Histological Nomenclature. This resulted in the editing of the Nomina Histologica Veterinaria: A Working Draft II (Toulouse, 1974), followed by preparations for publication of a Nomina Histologica Veterinaria.
    [Show full text]
  • Structure of Nucleoli in First-Order Spermatocytes of Selected Free
    G Model ANIREP-5223; No. of Pages 7 ARTICLE IN PRESS Animal Reproduction Science xxx (2015) xxx–xxx Contents lists available at ScienceDirect Animal Reproduction Science jou rnal homepage: www.elsevier.com/locate/anireprosci Structure of nucleoli in first-order spermatocytes of selected free-living animal species a,∗ b a Katarzyna Andraszek , Magdalena Gryzinska´ , Mariola Ceranka , a Agnieszka Larisch a Department of Animal Genetics and Horse Breeding, Institute of Bioengineering and Animal Breeding, University of Natural Sciences and Humanities, 14 Prusa Str, 08-110 Siedlce, Poland b Department of Biological Basis of Animal Production, University of Life Sciences, Lublin, Akademicka 13, 20-950 Lublin, Poland a r t a b i c l e i n f o s t r a c t Article history: Nucleoli are the product of the activity of nucleolar organizer regions (NOR) in certain chro- Received 23 February 2015 mosomes. Their main functions are the formation of ribosomal subunits from ribosomal Received in revised form 17 June 2015 protein molecules and the transcription of genes encoding rRNA. Nucleoli are present in Accepted 19 June 2015 the nuclei of nearly all eukaryotic cells because they contain housekeeping genes. The size Available online xxx and number of nucleoli gradually decrease during spermatogenesis. Some of the material originating in the nucleolus probably migrates to the cytoplasm and takes part in the for- Keywords: mation of chromatoid bodies (CB). Nucleolus fragmentation and CB assembly take place Roe deer at the same stage of spermatogenesis. CB are involved in the formation of the acrosome, Wild boar Spermatogenesis the migration of mitochondria to the midpiece, and the formation of the sperm tail fibrous Nucleolus sheath.
    [Show full text]