ISSN 0006-2979, Biochemistry (Moscow), 2016, Vol. 81, No. 10, pp. 1057-1065. © Pleiades Publishing, Ltd., 2016. Original Russian Text © A. A. Kolesnikov, 2016, published in Biokhimiya, 2016, Vol. 81, No. 10, pp. 1322-1331. REVIEW The Mitochondrial Genome. The Nucleoid A. A. Kolesnikov Lomonosov Moscow State University, Faculty of Biology, 119991 Moscow, Russia; E-mail: [email protected] Received May 30, 2016 Revision received July 1, 2016 Abstract—Mitochondrial DNA (mtDNA) in cells is organized in nucleoids containing DNA and various proteins. This review discusses questions of organization and structural dynamics of nucleoids as well as their protein components. The structures of mt-nucleoid from different organisms are compared. The currently accepted model of nucleoid organization is described and questions needing answers for better understanding of the fine mechanisms of the mitochondrial genetic apparatus functioning are discussed. DOI: 10.1134/S0006297916100047 Key words: mitochondrial nucleoid, mitochondrial genome It is now clear that understanding of genome organ- mtDNA). In fact, proteins associated with a nucleoid can ization of subcellular structures is necessary for under- influence the speed of accumulation of mutations. standing of cellular processes occurring through the cell In the last decade, reviews devoted to this subject cycle, including questions of storage, realization, and appeared regularly [5-9]. Generally, they cover the subject transmission of genetic information. A considerable of nucleoid organization in metazoan mitochondria amount of information about forms and sizes of mito- (mainly in human cells) and fungi (mainly yeast). chondrial DNA (mtDNA) throughout evolution has been Reviews touching plant cells are much less frequent [10, accumulated [1]. Since the discovery of mtDNA [2, 3], it 11]. This can be explained by more complex organization has been clear that organization of mtDNA differs signif- of the plant mt-genome and, first of all, by the extremely icantly from the organization of nuclear chromatin. For a big size of mtDNA (in some species, mtDNA is more long time, mtDNA was not supposed to be covered with than 1,000,000-bp-long); thus, more complicated exper- proteins, and some authors even used the term “naked” imental approaches are needed to analyze the organiza- DNA, which is not true. Up to now, the list of proteins tion of the mt-genome in this group of organisms. that can be associated with mtDNA in most characterized mt-nucleoids of human cells includes more than 50 items and continues to grow [4]. Classical methods of cellular NUCLEOID MORPHOLOGY biology allow analysis of nucleoid topology in a cell and the presence of certain components (proteins, RNA, etc.) Several words must be said about terminology. The in a nucleoid, but they are not able to provide detailed nucleoid is an area in a mitochondrion (similarly in information about specific features of nucleoid structure. chloroplasts) that contains DNA associated with proteins The main approach to studying nucleoids is to analyze necessary for the maintenance of mtDNA integrity, com- natural DNA–protein complexes. plexes involved in transfer of genetic information to It was recently suggested that the mitochondrial daughter cells and its realization. Although the nucleoid chromosome and not the mtDNA is a unit of inheritance is not separated from the matrix by a classical membrane, of a mitochondrial genome, which makes studies of one can expect that the nucleoid forms a separate com- nucleoid structure quite important. Moreover, deep inter- partment due to some substance. The nature of this sub- est in the nucleoid is stimulated by, so-called, “mito- stance remains unclear. The term nucleoid was proposed chondrial diseases” (diseases of multicellular organisms by analogy to prokaryotic cells. Some authors use this associated with accumulation of numerous mutations in term to designate only the DNA–protein complex (DNP), which is not completely correct in our opinion. Abbreviations: mtDNA, mitochondrial DNA; mt genome, Sometimes the term mitochondrial chromosome is used mitochondrial genome. instead of DNP. 1057 1058 KOLESNIKOV Direct experimental studies of nucleoid structure are yeast nucleoid contains 1-2 molecules of mtDNA, where- being done using several approaches: (i) by use of elec- as the mt-nucleoid of vertebrates usually contains 5-7 tron, fluorescence, and confocal microscopy [12, 13]; (ii) molecules [20, 21]. In recent years, the development of by analysis of DNP purified from isolated mitochondria; high-resolution microscopy adjusted this parameter for (iii) by genetic methods. Microscopy reveals the form and mammalian cells. It is now accepted that a nucleoid con- the size of a nucleoid and allows estimation of the colo- tains 1-2 mtDNA molecules in mammals [19]. At the calization of individual proteins with DNA. Nucleoid can same time, it was shown that although the main portion of be visualized using either fluorescent dyes (DAPI, mtDNA in mammalian cells is represented by single cir- PicoGreen, EtBr, etc.) [14, 15]; fluorescently labeled cular molecules, in mouse cells from different tissues antibodies to DNA or protein components of a nucleoid about 10% of mtDNA molecules form catenanes, and in [12, 16]; by means of expression of fluorescent fusion human NEK cell culture about 30%. It is clear that in this proteins (in human cells, this approach was used to local- case one nucleoid can contain more than 1-2 mtDNA ize TFAM or Twinkle proteins in DNP) [16, 17]; or by molecules [33, 34]. A similar situation is observed in means of expression of fluorescent photoactivated pro- embryonic cells, where the number of mtDNA molecules teins mEos2 or Dronpa [18]. The latter approach allows is 3-4 orders of magnitude higher than in somatic cells. In monitoring of the lifetime dynamics of a nucleoid. trypanosomatids, several thousand individual DNA mole- Mitochondrial nucleoids appear as spherical or ellipsoid cules are unified in a single complex associate that consti- structures of 70-100 nm diameter in human cells and of tutes a basis of one (unique) nucleoid. Concerning the 400 nm in yeast [6, 19]. In mitochondria of plant cells, number of mtDNA molecules composing a nucleoid, it is the spindle-shaped form of an mt-chromosome is probably more appropriate to consider not DNA mole- observed more often [10, 11]. A limited number of species cules, but the number of full-size mitochondrial genomes. was used for these studies: mammalian cells (generally How many nucleoids can one mitochondrion contain? human cell cultures), Xenopus oocytes, yeast, protozoa, Considering the dynamic nature of mitochondria, it is and higher plants (beans, tobacco cultured cells, etc.) difficult to answer this question. During the major part of [20-24]. It is important to mention that the number of a cell cycle, mitochondria are multinucleoid structures nucleoids in human cells varies from 500 to 12,900 (mt-reticulum). However, in synchronized culture of depending on the cell type and the method of nucleoid osteosarcoma (143B) cells, it was shown that mt-reticu- visualization (cited by [7]), in fibroblasts about 1000 lum is fragmented, and one nucleoid can be seen in a dis- nucleoids can be detected [7], in yeast – about 8-10, in crete mitochondrion [14]. An interesting fact was trypanosomatids – one complex associate [25]. The num- observed: if mitochondria were studied after mechanical ber of nucleoids per cell and not per mitochondrion is breakage of cells, a significant number of mitochondria calculated since in many cells mitochondria are not dis- (~40%) did not contain DNA (a nucleoid). crete small spherical bodies but have a form of “mito- chondrial network”, constantly changing its form because of the continuous fusion/fission process [19]. The mito- DNP COMPLEXES. PROTEINS chondrial network is often seen in young, actively divid- ASSOCIATED WITH mtDNA ing cells. Constant progress in microscopy techniques allowed obtaining images of individual DNA–protein The second and, in fact, the main approach for complexes with resolution to 10 nm. These techniques studying the molecular organization of nucleoids is to are, for example, STED microscopy, and PALM and analyze proteins, specifically bound to or interacting with STORM [19, 26-32]. Standard fluorescence microscopy mt-DNA (DNP). The strategy of studying nucleoid pro- allows obtaining resolution of 200-350 nm. teins consists of preparation of highly pure mitochondria STED (stimulated emission depletion) microscopy – a with subsequent purification of DNP complexes. modification of fluorescence microscopy enabling resolu- Sometimes, the size of a nucleoid is so big that it is possi- tion 30-150 nm below the diffraction limit. ble to purify it using a combination of differential and STORM (stochastic optical reconstruction gradient-density centrifugation [27, 35, 36]. microscopy) – resolution of individual molecules to 10- Electron microscopy shows that the purified sample 25 nm. consists of spherical particles with uniform mean size. PALM (photo activated localization microscopy) The main problem is to avoid reorganization (remodel- allows distinguishing individual molecules. ing) of the complex, which can happen during purifica- Data collected with these methods suggest that the tion of DNP. To exclude misinterpretation of the results, density of mt-chromosome packing excludes the diffu- the composition of native DNP and DNP fixed with sion of soluble proteins of mitochondrial