Proliferation without nuclei suggests mitochondrial cells (MitoCells) to be prokaryotic nature

Abstract

It is widely believed that evolutionarily advanced cells require nuclei rich in DNA to proliferate, while mitochondria isolated from nuclei cannot proliferate. We have developed a novel stable cell line, designated “mitochondrial cells (MitoCells)”, maintaining active mitochondria, the majority of which lacked nuclei. MitoCells can be continuously generated in culture. Herein, we report proliferation studies of MitoCells. We found that the nuclear DNA-less (nDNA-less) MitoCells, which were sorted with flow cytometry and were filtrated with 1.2 μmeter and 3 μmeter isopore membrane filters, could exist and proliferate. We also confirmed that the sorted nDNA-less MitoCells had mitochondrial DNA(mtDNA) with T9176C mutation analysis and that the unsorted MitoCells had SURF-1 gene of nDNA. These results suggest that MitoCells could have prokaryotic nature with both mtDNA and nDNA. Introduction The eukaryotes, by definition and in contrast to prokaryotes, have a nucleus, which contains most of the cell’s DNA, enclosed by double layered membranes [1]. The prokaryotes have no nucleus and have few or no organelles. Eukaryotes must have a nucleus and mitochondria in order to exist and proliferate with some exceptions [2-4]. Mitochondria which are isolated from nuclei are believed to be unable to exist or proliferate [5,6]. It is hypothesized that most of the ancestral mitochondrial genome was transferred to the nucleus as the symbiotic relationship between eukaryotes and ancestral mitochondria developed [7-9]. The respiratory chain /oxidative phosphorylation system (OPHOS) has five complexes, the polypeptides of which are encoded by mitochondrial DNA (mtDNA) and/or nuclear DNA (nDNA): only 13 polypeptides are encoded by mtDNA: complex I has 7 polypeptides encoded by mtDNA, complex II none, complex III one, complex IV three and complex V two. All other polypeptides of mitochondria are encoded by nDNA. We reported a series of novel stable cell lines, designated “mitochondrial cells (MitoCells)”, derived from cybrids obtained by fusing mitochondria-less HeLa cells with platelets [10,11]. All MitoCells have active mitochondria. The majority of these MitoCells lack nuclei but have a small amount of DNA, while the minority (less than 3 percent) have the same amount of DNA as cells with nuclei (nDNA-rich). We have recently reported that the MitoCells can survive under anaerobic condition and they could produce energy employing an anaerobic metabolic pathway, maintaining the electron transport enzyme activities, but losing the activity of tricarboxylic acid (TCA) cycle by means of mitochondrial enzyme assay and Western blot analyses [12]. This report suggests that energy production of MitoCells is far from the original cybrid cells which are eukaryote, although the MitoCells have enough polypeptides encoded by mtDNA and nDNA for maintaining their lives. Although most MitoCells have no nuclei, they can be continuously generated in culture. Therefore, it is important to know the feature of the nuclear DNA-less (nDNAless) MitoCells, which do not possess nucleus. Herein, we assessed the proliferation study and mtDNA analysis of the nDNA-less MitoCells, Materials MitoCell lines were obtained by transformation of cybrids derived by fusing mitochondria-less HeLa cells with platelets from Leigh syndrome patients and controls. The MitoCells from patients with Leigh syndrome, a subtype of mitochondrial encephalomyopathy, contain a pathogenic point mutation, T9176C, in their mtDNA [1315]. Our cell lines #1 and #2 were derived from heteroplasmic T9176C mutant cybrids, while cell line #3 represents wild type homoplasmy from control cybrids. The methods for establishment and culture of cell lines were described in detail in previous subscripts [10,11]. Methods MitoCell proliferation analysis with flow cytometer Sorting of MitoCell by flow cytometry: Two samples were taken from each MitoCell line. After adding 1 μM SYTO Green (Syto 16) (Molecular Probes, Inc. Eugene OR), the samples were left standing for 15 minutes. SYTO Green is a fluorescent dye that stains nucleic acids even in live cells [15]. The samples were gated and sorted by flow cytometry (Epics ALTRATM HyperSortTM, Beckman Coulter Inc., Miami, FL). The SYTO Green intensity range of the nucleus was defined based on the control cybrid data. Culture of the sorted cells and cell count: The sorted MitoCells, (nDNA-less MitoCells) were cultured in fresh RPMI 1640 medium Correspondence to: Kazutoshi Nakano, MD, Nakano Children’s Clinic, Medical Center Kamifukuoka, Fujimino-shi, Saitama, Japan, E-mail: knakano2005@yahoo.co.jp