Stable heteroplasmy at the single cell level is facilitated by inter-cellular exchange of mtDNA

Eukaryotic cells carry two genomes, nuclear (nDNA) and mitochondrial (mtDNA), which are ostensibly decoupled in their replication, segregation and inheritance. It is increasingly appreciated that heteroplasmy, the occurrence of multiple mtDNA haplotypes in a cell, plays an important biological role, but its features are not well understood. Accurately determining the diversity of mtDNA has been difficult, due to the relatively small amount of mtDNA in each cell (< 1% of the total DNA), the intercellular variability of mtDNA content and mtDNA pseudogenes (Numts) in nDNA. To understand the nature of heteroplasmy, we developed Mseek, a novel technique to purify and sequence mtDNA. Mseek yields high purity (> 90%) mtDNA and its ability to detect rare variants is limited only by sequencing depth, providing unprecedented sensitivity and specificity. Using Mseek, we confirmed the ubiquity of heteroplasmy by analyzing mtDNA from a diverse set of cell lines and human samples. Applying Mseek to colonies derived from single cells, we find heteroplasmy is stably maintained in individual daughter cells over multiple cell divisions. We hypothesized that the stability of heteroplasmy could be facilitated by inter-cellular exchange of mtDNA. We explicitly demonstrate this exchange by co-culturing cell lines with distinct mtDNA haplotypes. Our results shed new light on the maintenance of heteroplasmy and provide a novel platform to investigate features of heteroplasmy in normal and diseased states.

[1]  Ernesto Picardi,et al.  MToolBox: a highly automated pipeline for heteroplasmy annotation and prioritization analysis of human mitochondrial variants in high-throughput sequencing , 2014, Bioinform..

[2]  Jian Lu,et al.  Extensive pathogenicity of mitochondrial heteroplasmy in healthy human individuals , 2014, Proceedings of the National Academy of Sciences.

[3]  A. Clark,et al.  Balancing Selection in Species with Separate Sexes: Insights from Fisher’s Geometric Model , 2014, Genetics.

[4]  Marcella Attimonelli,et al.  Extraction and annotation of human mitochondrial genomes from 1000 Genomes Whole Exome Sequencing data , 2014, BMC Genomics.

[5]  K. McKernan,et al.  Expanded Genetic Codes in Next Generation Sequencing Enable Decontamination and Mitochondrial Enrichment , 2014, PloS one.

[6]  D. Sabatini,et al.  Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides , 2014, Nature.

[7]  Sathish Kumar Mungamuri,et al.  p53-dependent gene repression through p21 is mediated by recruitment of E2F4 repression complexes , 2013, Oncogene.

[8]  J. Bhattacharya,et al.  When cells become organelle donors. , 2013, Physiology.

[9]  D. Wallace,et al.  Mitochondrial DNA genetics and the heteroplasmy conundrum in evolution and disease. , 2013, Cold Spring Harbor perspectives in biology.

[10]  J. Vijg,et al.  Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing. , 2013, BioTechniques.

[11]  Ravi Sachidanandam,et al.  MiST: A new approach to variant detection in deep sequencing datasets , 2013, Nucleic acids research.

[12]  Y. Bouhlal,et al.  Twin mitochondrial sequence analysis , 2013, Molecular genetics & genomic medicine.

[13]  D. Egli,et al.  Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants , 2012, Nature.

[14]  Carolina Wählby,et al.  Non-Random mtDNA Segregation Patterns Indicate a Metastable Heteroplasmic Segregation Unit in m.3243A>G Cybrid Cells , 2012, PloS one.

[15]  Eitan Rubin,et al.  Mitochondrial DNA heteroplasmy in diabetes and normal adults: role of acquired and inherited mutational patterns in twins. , 2012, Human molecular genetics.

[16]  Lynda Chin,et al.  Spectrum of somatic mitochondrial mutations in five cancers , 2012, Proceedings of the National Academy of Sciences.

[17]  Jesse J. Salk,et al.  Detection of ultra-rare mutations by next-generation sequencing , 2012, Proceedings of the National Academy of Sciences.

[18]  G. Pesole,et al.  Mitochondrial genomes gleaned from human whole-exome sequencing , 2012, Nature Methods.

[19]  D. Rowlands,et al.  Mitochondrial transfer from bone-marrow–derived stromal cells to pulmonary alveoli protects against acute lung injury , 2012, Nature Medicine.

[20]  Marcella Attimonelli,et al.  Primates and mouse NumtS in the UCSC Genome Browser , 2012, BMC Bioinformatics.

[21]  P. Vallone.  Capillary electrophoresis of an 11-plex mtDNA coding region SNP single base extension assay for discrimination of the most common Caucasian HV1/HV2 mitotype. , 2012, Methods in molecular biology.

[22]  C. Sander,et al.  Predicting the functional impact of protein mutations: application to cancer genomics , 2011, Nucleic acids research.

[23]  Anton Nekrutenko,et al.  Dynamics of mitochondrial heteroplasmy in three families investigated via a repeatable re-sequencing study , 2011, Genome Biology.

[24]  Max Ingman,et al.  Ultra-Deep Sequencing of Mouse Mitochondrial DNA: Mutational Patterns and Their Origins , 2011, PLoS genetics.

[25]  J. Enríquez,et al.  Tissue-specific differences in mitochondrial activity and biogenesis. , 2011, Mitochondrion.

[26]  S. Pääbo,et al.  Multiplexed DNA Sequence Capture of Mitochondrial Genomes Using PCR Products , 2010, PloS one.

[27]  T. Prolla,et al.  Mitochondrial Fusion Is Required for mtDNA Stability in Skeletal Muscle and Tolerance of mtDNA Mutations , 2010, Cell.

[28]  D. Dressman,et al.  Heteroplasmic mitochondrial DNA mutations in normal and tumor cells , 2010, Nature.

[29]  W. Junger,et al.  Circulating Mitochondrial DAMPs Cause Inflammatory Responses to Injury , 2009, Nature.

[30]  U. Friberg,et al.  Evolutionary implications of non-neutral mitochondrial genetic variation. , 2008, Trends in ecology & evolution.

[31]  M. Attimonelli,et al.  The RHNumtS compilation: Features and bioinformatics approaches to locate and quantify Human NumtS , 2008, BMC Genomics.

[32]  B. Lang,et al.  Purification of mitochondrial and plastid DNA , 2007, Nature Protocols.

[33]  Laura C. Greaves,et al.  Mitochondrial DNA mutations in human disease , 2006, IUBMB life.

[34]  Darwin J. Prockop,et al.  Mitochondrial transfer between cells can rescue aerobic respiration , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Shamkant B. Navathe,et al.  MITOMAP: a human mitochondrial genome database—2004 update , 2004, Nucleic Acids Res..

[36]  S. Osawa,et al.  The genetic code in mitochondria and chloroplasts , 1990, Experientia.

[37]  P. Frachon,et al.  Organization and dynamics of human mitochondrial DNA , 2004, Journal of Cell Science.

[38]  P. Bénit,et al.  Recurrent de novo mitochondrial DNA mutations in respiratory chain deficiency , 2003, Journal of medical genetics.

[39]  Benedikt Westermann,et al.  'Omics' of the mitochondrion , 2003, Nature Biotechnology.

[40]  D. Newmeyer,et al.  Mitochondria Releasing Power for Life and Unleashing the Machineries of Death , 2003, Cell.

[41]  M. Woischnik,et al.  Pattern of organization of human mitochondrial pseudogenes in the nuclear genome. , 2002, Genome research.

[42]  H. Coller,et al.  High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection , 2001, Nature Genetics.

[43]  D. Clayton.  Transcription and replication of mitochondrial DNA. , 2000, Human reproduction.

[44]  J. Piškur,et al.  Horizontal Transfer of Genetic Material amongSaccharomyces Yeasts , 1999, Journal of bacteriology.

[45]  D. Wallace.  Mitochondrial diseases in man and mouse. , 1999, Science.

[46]  Xiaodong Wang,et al.  Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome c , 1996, Cell.

[47]  D. Wallace.  Mitochondrial DNA mutations in diseases of energy metabolism , 1994, Journal of bioenergetics and biomembranes.

[48]  D. C. Wallace.  Structure and evolution of organelle genomes. , 1982, Microbiological reviews.