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Analysis of Human Mitochondrial Transcripts Using Electron Microscopic in Situ Hybridization

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Analysis of Human Mitochondrial Transcripts Using Electron Microscopic in Situ Hybridization Analysis of human mitochondrial transcripts using electron microscopic in situ hybridization FRANCOISE ESCAIG-HAYE1'2, VLADIMIR GRIGORIEV3, GABRIEL PERANZI4, PATRICK LESTIENNE5 and JEAN-GUY FOURNIER12 1INSERM Uniti 43, Hdpital St Vincent de Paul, 74 avenue Denfert-Rochereau, 75674 Paris Cedex 14, France 2INSERM Unit6 153, 17 rue du Fer-a-Moulin, 75005 Paris, France 3Iuanousky Institute, Gamaleya Street, Moscow, USSR 4INSERM UnM 10, Hdpital Bichat, 170 boulevard Ney, 75877 Paris Cedex 18, France 5INSERM Unite 298, CHR, 49033 Angers Cedex, France Summary Human mitochondrial transcripts have been exam- analysis indicates that the mitochondria concentrate ined at the ultrastructural level. After contact with the labeling with intensities that vary with the type of ultrathin sections of a human lymphoid cell line RNA and that the nucleus induces a light hybridiz- (CEM) embedded in Lowicryl K4M, biotinylated ation signal with each mitochondrial probe. The mitochondrial probes yield specific hybrids ident- visualization of human mitochondrial DNA ex- ified by a colloidal gold immunocytochemistry pression in correlation with the fine anatomy of the marker that visualizes rRNA and mRNA coding for mitochondria constitutes a new approach for funda- respiratory chain polypeptides CO II, CO III and mental research on the organelle and for analyzing ATPase-6. The mitochondrial transcripts are prefer- its behaviour in human mitochondrial diseases. entially located close to the inner membrane, particu- larly the cristae, suggesting that intra-organelle protein synthesis is intimately associated with the Key words: mitochondria, transcription, ultrastructure, mitochondrial membrane system. Quantitative hybridization. Introduction and function, since the morphological organization of mitochondria is visible uniquely under the electron The mitochondria occupy a unique situation among the microscope. Recently, several attempts have been made to cellular organelles because they play a major role in the adapt an in situ hybridization protocol to electron cell respiratory function and this function requires microscopy. Among the different approaches, those using coordinated interaction of the nuclear and the mitochon- probes non-isotopically labeled with biotin (Langer et al. drial genomes. The recent application of recombinant 1981; Singer and Ward, 1982; Singer et al. 1987) to perform DNA technology to the human mitochondrial genome has hybridization either before (Wolber et al. 1989; Silva et al. permitted the determination of its complete sequences and 1989; Singer et al. 19896), after (Binder et al. 1986; to observe the extreme compactness of its organization Webster et al. 1987; Brangeon et al. 1989; Escaig-Haye et (Anderson et al. 1981). The heavy strand contains the al. 1989; Thiry and Thiry-Blaise, 1989) or without (Radic et genes for two rRNAs, 14 tRNAs and 12 reading frames, al. 1987; Singer et al. 1989a) cell embedding in resin, offer while the light strand contains the genes for eight tRNAs a very rapid and effective method for the detection of the and one reading frame. The two strands are transcribed in nucleic acid integrated in their sub-cellular environment. the form of polycistronic molecules that are processed by In this work, the method of approach is to hybridize endonucleotide cleavage to yield the individual mature ultra-thin sections of material embedded in the hydrosol- mRNA species necessary for the synthesis of 13 polypep- uble resin Lowicryl K4M with a biotinylated DNA probe, tide components of the respiratory chain (Murphy et al. then, using colloidal gold immunocytochemistry marker 1975; Qjala et al. 1981; Bogenhagen et al. 1984; Chang and technology, to reveal the site of the specific hybrid Clayton, 1984; Shuey and Attardi, 1985; Kruse et al. 1989). formation. We have previously employed this protocol for To gain insight into the genetic functions of human detecting cellular RNA (Escaig-Haye et al. 1989) and a mitochondria required for oxidative phosphorylation, it similar approach has been used to detect ribosomal appeared essential to examine the mitochondrial tran- mitochondrial RNA in Drosophila cells (Binder et al. scripts in relation to the fine structure of the organelle's 1986). In the present case, expression of the human morphology. In that case, ultrastructural detection of mitochondrial genome was examined in a lymphoblastoid nucleic acid molecules is the only means of examining the human cell line with three different mitochondrial probes, molecular aspect of the relationship between structure one detecting ribosomal RNA, another detecting simul- Journal of Cell Science 100, 851-862 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 851 taneously two mRNA populations corresponding to cyto- floating the grids on drops of the hybridization solution 1 chrome c oxidase subunit III (CO III) mRNA and ATPase containing the DNA probes at a concentration of 10-20 ng ml" in subunit 6 (ATPase-6) mRNA, and the last one detecting a moist chamber for 16 h at 37 °C. The grids were then washed cytochrome c oxidase subunit II (CO II) mRNA. successively in 50% formamide, 4xSSC (twice for 5min), 4xSSC (twice for 5 min), 0.2xSSC (twice for 5 min) and distilled water Qualitative analysis of the results indicated that the (once for 2 min). mitochondrial RNA was mainly localized close to the inner membrane of the mitochondria, particularly the cristae; Immunocytochemistry this was confirmed by quantitative analysis, which also showed that very different amounts of molecules were After hybridization washings, grids were incubated in PBS buffer containing 1 % bovine serum albumin (PBS/BSA, 1 %) for 15 min. detected according to the probe used. Several controls were Then the sections were incubated with rabbit antibiotin (Enzo, performed to confirm the specificity of the data and Biochemical) diluted at 1 % in PBS/BSA (1 %) for 60 min. After statistical analysis indicated that the nucleus reacted with incubation with the antibody, grids were rinsed in PBS buffer the mitochondrial probes. containing 0.05% Tween 20 (PBS/Tween 20, 0.05%) twice for 10 min and then were kept in contact for 60 min with goat anti- rabbit antibody complexed with 10 nm gold particles (Janssen, Materials and methods Belgium) diluted at 1/50 in PBS/BSA (1%) solution. Sub- sequently, grids were rinsed in PBS/Tween 20, 0.05% and Materials distilled water twice for 10 min each. Sections were next stained with uranyl acetate for 20 min and rinsed in distilled water twice The CEM lymphoblastoid cell line was maintained in suspension for 5 min. They were observed in a CM10 Philips electron in RPMI medium supplemented with 10% fetal calf serum, microscope at an acceleration voltage of 60 kV. streptamicin and penicillin. Human peripheral blood mono- nuclear cells were purified on a Ficoll-Hypaque gradient and stimulated for 3 days with the same medium containing PHA Quantitation analysis of labeling density (lO/igml"1). Human tissues were obtained from lymph-node and The labeling densities over various cell compartments of the CEM muscle biopsies and from a surgically removed appendix. cells were evaluated on negative electron micrographs taken at a minimal magnification of x 11500. A total of 15-25 electron micrographs were recorded for each sample. The surface areas Cells and tissue processing 3 occupied by the mitochondria and nucleus were evaluated using Cell pellets and 1 mm pieces of tissue were fixed by immersion for an image-analysing system, Mode Biocom 200. The surface area 2 h either in 4 % paraformaldehyde and 0.1% glutaraldehyde or in of the cytoplasm was obtained by subtracting the mitochondria 2 % paraformaldehyde and 0.2 % glutaraldehyde, diluted in 0.1 M and nucleus areas from the total surface of the negative electron sodium phosphate buffer (pH7.4), and then rinsed in the buffer micrographs. The number of gold particles present over each of twice for 5 min each. The biological material could then be stored the compartments was counted manually and the density of in the buffer complemented with sucrose for several days at 4°C. labeling was calculated by dividing the number of particles by the Embedding in Lowicryl was done according to Bendayan (1984), surface area. The results are expressed as the mean number of as previously described (Grigoriev et al. 1989; Escaig-Haye et al. gold particles^m~2±standard deviation of different samples. 1989). Ultrathin sections (60-80 nm) were cut on a Reichert Ultracut ultramicrotome and sections were collected directly on Statistical analysis was performed by Student's i-test. acetone-treated naked gold grids (hexagonal 600 mesh). Grids were kept at room temperature for no more than 3 weeks before use. Results Probes and nick-translation To analyse the human mitochondrial transcripts at the The mitochondrial probes consist of three different fragments of ultrastructural level, probes specific for different mito- the DNA genome. The first corresponds to 1.5 kb of Xbal 1-2 chondrial genes were hybridized to ultrathin sections of a fragment (1193-2953) containing the 12 S and 16 S ribosomal human lymphoid cell line (CEM) embedded in the gene sequences inserted into pUC19 (pMT rRNA). The second is hydrosoluble resin Lowicryl K4M. Using this cell material the Xbal 3-4 fragment (7440-8286) containing the CO II gene in enabled us to observe the good preservation of the pUC19 (pMT CO II) and the third, the Mbol fragment structure of many mitochondria, permitting us to clearly (8729-10 254)
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