Functional Constraints of Nuclear-Mitochondrial DNA Interactions in Xenomitochondrial Rodent Cell Lines

Abstract

The co-evolution of nuclear and mitochondrial genomes in vertebrates led to more than 100 specific interactions that are crucial for an optimized ATP generation. These interactions have been examined by introducing rat mtDNA into mouse cells devoid of mitochondrial DNA (mtDNA). When mtDNA-less cells derived from the common mouse (Mus musculus domesticus) were fused to cytoplasts prepared from Mus musculus, Mus spretus, or rat (Rattus norvegicus), a comparable number of respiring clones could be obtained. Mouse xenomitochondrial cybrids harboring rat mtDNA had a slower growth rate in medium containing galactose as the carbon source, suggesting a defect in oxidative phosphorylation. These clones respired approximately 50% less than the parental mouse cells or xenomitochondrial cybrids harboring Mus spretus mtDNA. The activities of respiratory complexes I and IV were approximately 50% lower, but mitochondrial protein synthesis was unaffected. The defects in complexes I and IV were associated with decreased steady-state levels of respective subunits suggesting problems in assembly. We also showed that the presence of 10% mouse mtDNA co-existing with rat mtDNA was sufficient to restore respiration to normal levels. Our results suggest that evolutionary distance alone is not a precise predictor of nuclear-mitochondrial interactions as previously suggested for primates.Nuclear-mitochondrial interactions play a fundamental role in cellular homeostasis. The nuclear genome encodes more than 95% of all proteins located in the mitochondria, whereas only 13 polypeptides (all subunits of the oxidative phosphorylation system, OXPHOS) are encoded by the mitochondrial genome. An optimal interaction between nuclear and mitochondrial encoded factors is essential for transcription and translation of mitochondrial DNA (mtDNA) 1 and also for the correct assembly and function of the OXPHOS system (1). Various attempts have been made to understand the interactions between the nuclear and mitochondrial genomes and their respective contributions to the expression of different phenotypes such as tumorigenicity (2, 3) and cell differentiation (4, 5). One approach that has facilitated the study of nuclear-mitochondrial interactions is the construction of interspecific hybrids and cybrids. Because the mtDNA sequence divergence is about 5-13 times more rapid than in nuclear DNA (nDNA), a general incompatibility between nuclear and mitochondrially coded gene products is expected between pairs of even recently diverged taxa (6).Previously, Kenyon and Moraes (7) demonstrated that OX-PHOS function of a human cell line lacking mtDNA ( 0 ) could be restored by inserting mitochondria from other humanoid primates including common chimpanzee (Pan troglodytes), pigmy chimpanzee (Pan paniscus), or gorilla (Gorilla gorilla). These studies suggested that at least in primates, mitochondrial/nuclear compatibility has been retained over approximately 5-12 million years (Myr) period. On the other hand, mtDNA from orangutan (Pongo pygmaeus), a species that div...

Interspecies mitochondrial fusion between mouse and human mitochondria is rapid and efficient

“…Interspecies somatic hybrid cells between mouse and human have been obtained, but the maintenance of mitochondrial genomes, which are usually from only one species, has been shown to require essentially a complete set of cognate chromosomes (Wallace et al, 1976;De Francesco et al, 1980). Viable xenomitochondrial cybrids between cells from different rodent species (e.g., mouse and rat) (Dey et al, 2000;McKenzie and Trounce, 2000;Yamaoka et al, 2000;McKenzie et al, 2003) or human-humanoid primates (e.g., human and chimpanzee or gorilla or orangutan) (Kenyon and Moraes, 1997;Bayona-Bafaluy et al, 2005) have been reported, and transmitochondrial or xenomitochondrial mice have been produced (Pinkert and Trounce, 2000;Shoubridge, 2000;Sokolova et al, 2004), but these studies also suggest that the retention of nuclear-mitochondrial compatibility between species is essential for generating interspecies xenomitochondrial cybrids or animal models with a functional (but reduced) oxidative phosphorylation system. Following intraspecies fusion of eukaryotic cells, the mitochondrial networks of the two cells rapidly fuse and the molecular contents of these networks are exchanged (Nunnari et al, 1997;Legros et al, 2002).…”

The consequences of nuclear transfer for mammalian foetal development and offspring survival. A mitochondrial DNA perspective

“…Table 2 clearly highlights the variation in mtDNA replication and transcription that can arise through nucleo-cytoplasmic variation. Of specific interest are the results arising from interspecies cybrid generation where rat mtDNA is efficiently replicated, transcribed, and translated against a murine nuclear background, although OXPHOS function is compromised, which compares unfavourably with murine -murine cybrids (Dey et al 2000, McKenzie & Trounce 2000, McKenzie et al 2003. This is further exemplified as primate mtDNA is only replicated when the human cell's own mtDNA has been eliminated (Moraes et al 1999).…”

“…More recently, the fate of mtDNA populations against different nuclear backgrounds have been analysed (Dunbar et al 1995, Barrientos et al 1998, Moraes et al 1999, Dey et al 2000, McKenzie & Trounce 2000, McKenzie et al 2003. Typically, a homoplasmic cybrid is formed by the fusion of an enucleated cell (cytoplast) with an mtDNA-depleted cell possessing a somatic nucleus (karyoplast).…”

“…MtDNA, NT and mammalian development 635 Dey et al 2000). Cybrid technology has also been used to analyse the transmission of mtDNA to offspring by introducing mtDNA into one-cell embryos (Inoue et al 2000) and also into blastocysts (Sligh et al 2000).…”

Identification of differential transcript profiles between mutual crossbred embryos of zebrafish (Danio rerio) and Chinese rare minnow (Gobiocypris rarus) by cDNA-AFLP

“…In cross-species NT embryos, whether heteroplasmic or not, incompatibility between the nuclear and cytoplasmic counterpart may be partially responsible for the developmental arrest [26], and the interaction of mitochondria from one species with the nucleus of an unrelated species may result in serious metabolic disruption [26][27][28]. In accordance with these, we identified five differentially expressed genes (ZR2, ZR3, ZR9, RZ3, and RZ6) related to metabolism.…”