scholarly journals Modulation of mitochondrial nucleoid structure during aging and by mtDNA content in Drosophila

Biology Open ◽  
2021 ◽  
Vol 10 (6) ◽  
Author(s):  
Li-jie Wang ◽  
Tian Hsu ◽  
Hsiang-ling Lin ◽  
Chi-yu Fu
Keyword(s):  
Flight Muscle ◽  
Ultrastructural Level ◽  
Gene Products ◽  
Mitochondrial Nucleoid ◽  
Copy Numbers ◽  
Drosophila Model ◽  
Nucleoid Structure ◽  
Mtdna Transcription ◽  
Transcription And Replication

ABSTRACT Mitochondrial DNA (mtDNA) encodes gene products that are essential for oxidative phosphorylation. They organize as higher order nucleoid structures (mtNucleoids) that were shown to be critical for the maintenance of mtDNA stability and integrity. While mtNucleoid structures are associated with cellular health, how they change in situ under physiological maturation and aging requires further investigation. In this study, we investigated the mtNucleoid assembly at an ultrastructural level in situ using the TFAM-Apex2 Drosophila model. We found that smaller and more compact TFAM-nucleoids are populated in the mitochondria of indirect flight muscle of aged flies. Furthermore, mtDNA transcription and replication were cross-regulated in the mtTFB2-knockdown flies as in the mtRNAPol-knockdown flies that resulted in reductions in mtDNA copy numbers and nucleoid-associated TFAM. Overall, our study reveals that the modulation of TFAM-nucleoid structure under physiological aging, which is critically regulated by mtDNA content.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

Cell cycle redistribution of U3 snRNA and fibrillarin. Presence in the cytoplasmic nucleolus remnant and in the prenucleolar bodies at telophase

1994 ◽  
Vol 107 (2) ◽  
pp. 463-475 ◽  
Author(s):  
M.C. Azum-Gelade ◽  
J. Noaillac-Depeyre ◽  
M. Caizergues-Ferrer ◽  
N. Gas
Keyword(s):  
Cell Cycle ◽  
Immunofluorescence Microscopy ◽  
Close Association ◽  
Ultrastructural Level ◽  
Small Nuclear Rna ◽  
Cho Cell ◽  
Nucleolar Proteins ◽  
Nuclear Rna ◽  
Active Nors

The distribution of the U3 small nuclear RNA during the cell cycle of the CHO cell line was studied by in situ hybridization using digoxigenin-labelled oligonucleotide probes. The location of the hybrids by immunofluorescence microscopy and at the ultrastructural level was correlated with the distribution of two nucleolar proteins, nucleolin and fibrillarin. The U3 snRNA molecules persist throughout mitosis in close association with the nucleolar remnant. U3 snRNA is present in the prenucleolar bodies (PNBs) and could participate in nucleologenesis in association with several nucleolar proteins such as nucleolin and fibrillarin. The interaction of U3 snRNP with the 5′ external spacer of pre-RNA newly synthesized by active NORs is proposed to be the promoting event of nucleologenesis.

scholarly journals Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
William A Kronert ◽  
Kaylyn M Bell ◽  
Meera C Viswanathan ◽  
Girish C Melkani ◽  
Adriana S Trujillo ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Flight Muscle ◽  
Isometric Force ◽  
Muscle Mechanics ◽  
Muscle Dysfunction ◽  
Cardiac Myosin ◽  
Cross Bridge ◽  
Drosophila Model ◽  
Mechanistic Basis ◽  
Tension Generation

K146N is a dominant mutation in human β-cardiac myosin heavy chain, which causes hypertrophic cardiomyopathy. We examined how Drosophila muscle responds to this mutation and integratively analyzed the biochemical, physiological and mechanical foundations of the disease. ATPase assays, actin motility, and indirect flight muscle mechanics suggest at least two rate constants of the cross-bridge cycle are altered by the mutation: increased myosin attachment to actin and decreased detachment, yielding prolonged binding. This increases isometric force generation, but also resistive force and work absorption during cyclical contractions, resulting in decreased work, power output, flight ability and degeneration of flight muscle sarcomere morphology. Consistent with prolonged cross-bridge binding serving as the mechanistic basis of the disease and with human phenotypes, 146N/+ hearts are hypercontractile with increased tension generation periods, decreased diastolic/systolic diameters and myofibrillar disarray. This suggests that screening mutated Drosophila hearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments.

scholarly journals Immunodetection of RNA on ultra-thin sections incubated with polyadenylate nucleotidyl transferase.

1993 ◽  
Vol 41 (5) ◽  
pp. 657-665 ◽  
Author(s):  
M Thiry
Keyword(s):  
Divalent Cations ◽  
Ultrastructural Level ◽  
Ehrlich Tumor ◽  
Precise Location ◽  
Great Precision ◽  
Thin Sections ◽  
Nucleotidyl Transferase ◽  
Ehrlich Tumor Cells ◽  
Labeling Pattern

A new method is described for locating RNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing polyadenylate nucleotidyl transferase (PnT) and biotinylated ATP. The labeled nucleotides bound to RNA at the surface of the ultra-thin sections were than visualized by an indirect immunogold labeling technique. The resulting labeling pattern was dependent on the presence of divalent cations in the PnT medium. The method revealed with great precision the specific RNA-containing structures within Ehrlich tumor cells. The method is applicable to Epon sections. However, the labeling intensity varies according to the fixation used. Best results were obtained on acetylated cell sections. The method can be combined with EDTA regressive staining. The in situ PnT method provides a very useful tool for pinpointing the precise location of RNA within biological material at the ultrastructural level.

scholarly journals A Novel Flat-embedding Method to Prepare Ultrathin Cryosections from Cultured Cells in Their In Situ Orientation

2002 ◽  
Vol 50 (8) ◽  
pp. 1067-1080 ◽  
Author(s):  
Viola Oorschot ◽  
Heidi de Wit ◽  
Wim G. Annaert ◽  
Judith Klumperman
Keyword(s):  
Electron Microscopy ◽  
Quantitative Method ◽  
Cultured Cells ◽  
Petri Dish ◽  
Ultrastructural Level ◽  
Immunogold Labeling ◽  
Polarized Cells ◽  
Ultrathin Cryosections ◽  
Flat Embedding

Immunogold labeling of ultrathin cryosections provides a sensitive and quantitative method to localize proteins at the ultrastructural level. An obligatory step in the routine preparation of cryosections from cultured cells is the detachment of cells from their substrate and subsequent pelleting. This procedure precludes visualization of cells in their in situ orientation and hampers the study of polarized cells. Here we describe a method to sample cultured cells from a petri dish or coverslip by embedding them in a 12% gelatin slab. Subsequently, sections can be prepared in parallel or perpendicular to the plane of growth. Our method extends the cryosectioning technique to applications in studying polarized cells and correlative light–electron microscopy.

Localization of actin in the Tetrahymena basal body-cage complex

1992 ◽  
Vol 103 (3) ◽  
pp. 629-641 ◽  
Author(s):  
J.G. Hoey ◽  
R.H. Gavin
Keyword(s):  
Basal Body ◽  
Specific Binding ◽  
Ultrastructural Level ◽  
Basal Bodies ◽  
Muscle Actin ◽  
Cage Complex ◽  
Contractile System ◽  
Chicken Muscle ◽  
Filament Bundles

In the ciliate cytoskeleton, basal bodies are contained within separate, filamentous cages which are closely associated with basal body microtubules. We have used two polyclonal anti-actin antibodies to localize actin within the basal body-cage complex of Tetrahymena. An antiserum against a Tetrahymena oral apparatus fraction enriched for basal body proteins was produced in rabbits. Agarose-linked chicken muscle actin was used to affinity-purify anti-Tetrahymena actin antibodies from the anti-oral apparatus antiserum. Agarose-linked chicken muscle actin was used to affinity-purify anti-chicken muscle actin antibodies from a commercially available antiserum against chicken muscle actin. Both affinity-purified antibodies were monospecific for Tetrahymena actin on immunoblots containing total oral apparatus protein. The anti-actin antibodies were localized to both somatic and oral basal bodies in Tetrahymena by immunofluorescence microscopy. At the ultrastructural level with the immunogold technique, these antibodies labeled actin epitopes in four distinct regions of the basal body-cage complex: (a) basal body walls, (b) basal plate filaments, (c) proximal-end filaments and (d) cage wall filaments. In addition, the antibody labeled filament bundles that interconnect groups of basal bodies (membranelles) within the oral apparatus. Identical labeling patterns were observed with basal bodies in the isolated oral apparatus, basal bodies in the in situ oral apparatus and somatic basal bodies in situ. Quantitative analysis of gold particle distribution was used to demonstrate the specificity of the antibodies for the basal body-cage complex and to show that non-specific binding of the antibodies was negligible. Preadsorption of the antibody with muscle actin effectively eliminated the capacity of the antibody to bind to proteins on immunoblots and to basal body structures with the immunogold labeling technique. These results provide evidence for actin in the basal body-cage complex and raise the possibility of a contractile system associated with basal bodies.

scholarly journals ALK gene products in anaplastic large cell lymphomas and Hodgkin's disease

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1694-1700 ◽  
Author(s):  
H Herbst ◽  
J Anagnostopoulos ◽  
B Heinze ◽  
H Durkop ◽  
M Hummel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Hodgkin's Disease ◽  
Fusion Gene ◽  
Hodgkin’S Disease ◽  
Young Patients ◽  
Large Cell ◽  
Common Type ◽  
Gene Products ◽  
Cytogenetic Analyses

The translocation t(2;5)(p23;q35), discovered in CD30+ anaplastic large cell (ALC) lymphomas, creates a potentially oncogenic fusion gene, part of which is contributed by a novel tyrosine kinase, ALK. Absence of ALK expression from normal hematolymphoid cells provides a basis for the morphologic assessment of t(2;5). The distribution of the t(2;5) in ALC lymphomas and Hodgkin's disease (HD), as assayed by nonmorphologic methods, is controversial. We used in situ hybridization and/or immunohistology to show ALK gene products in 85 ALC lymphomas, 82 HD cases, 40 other lymphoproliferations, as well as in 6 HD- and 4 ALC lymphoma-derived cell lines. ALK gene products were restricted to t(2;5)-positive ALC lymphoma cell lines and tumor cells of 16 primary non-B cell, common-type ALC lymphomas. These were mainly from young patients with initial lymphonodal disease. ALK expression was not detectable in any other specimen, including all cases of HD and HD-like type ALC lymphoma as well as secondary ALC lymphomas. Full congruence was noted for labeling results obtained with both methods. In agreement with cytogenetic analyses, but at variance with recently published studies, ALK gene expression distinguishes a subset of ALC lymphomas from other CD30+ lymphomas, including HD. The results do not support concepts attributing a significant role to the t(2;5) in the development of HD.

Sign in / Sign up

Export Citation Format

Share Document