Stable retention of chloramphenicol-resistant mtDNA to rescue metabolically impaired cells

Abstract

The permanent transfer of specific mtDNA sequences into mammalian cells could generate improved models of mtDNA disease and support future cell-based therapies. Previous studies documented multiple biochemical changes in recipient cells shortly after mtDNA transfer, but the long-term retention and function of transferred mtDNA remains unknown. Here, we evaluate mtDNA retention in new host cells using 'MitoPunch', a device that transfers isolated mitochondria into mouse and human cells. We show that newly introduced mtDNA is stably retained in mtDNA-deficient (ρ0) recipient cells following uridine-free selection, although exogenous mtDNA is lost from metabolically impaired, mtDNA-intact (ρ+) cells. We then introduced a second selective pressure by transferring chloramphenicol-resistant mitochondria into chloramphenicol-sensitive, metabolically impaired ρ+ mouse cybrid cells. Following double selection, recipient cells with mismatched nuclear (nDNA) and mitochondrial (mtDNA) genomes retained transferred mtDNA, which replaced the endogenous mutant mtDNA and improved cell respiration. However, recipient cells with matched mtDNA-nDNA failed to retain transferred mtDNA and sustained impaired respiration. Our results suggest that exogenous mtDNA retention in metabolically impaired ρ+ recipients depends on the degree of recipient mtDNA-nDNA co-evolution. Uncovering factors that stabilize exogenous mtDNA integration will improve our understanding of in vivo mitochondrial transfer and the interplay between mitochondrial and nuclear genomes.

Figure 1

Stable mitochondrial integration in ρ0 cells. (a) Schematic showing selection of ρ0 cell with successfully retained exogenous mtDNA. (b) 143BTK- ρ0 cells with transferred HEK293T or MELAS A3243G mitochondria were selected on uridine-deficient media. Approximately 2 weeks after mitochondrial transfer, colonies were imaged on an inverted microscope and 5 × objective. (c) 143BTK- ρ0 + dsRed- labeled HEK293T mitochondria were visualized by DIC and fluorescence microscopy 1 and 2 weeks after mitochondrial transfer. (d) Sanger sequencing of 3 clones derived from 143BTK− ρ0 cells transferred HEK293T or MELAS mitochondria. Orange highlight denotes mtDNA position 3243. (e) Seahorse Extracellular Flux analysis to quantify oxygen consumption rate of bulk culture generated from 143BTK− ρ0 cells transferred HEK293T or MELAS mitochondria. (f) Seahorse Extracellular Flux analysis to quantify oxygen consumption rate of clones generated from (e). (e, f) Oligomycin, FCCP, and rotenone/myxothiazol are an ATP synthase inhibitor, uncoupler, and complex I/III inhibitors, respectively. Each data point represents the average of 3 technical replicates and the error bar denotes standard deviation.

Figure 2

Transfer of functional mtDNA is not maintained in ρ + mutant cells. (a) Schematic showing selection of ρ + mutant cell with transferred exogenous mtDNA. (b,c) Isolated dsRed-labeled HEK293T mitochondria were transferred by MitoPunch into MELAS cybrid cells and immediately analyzed by ImageStream. Brightfield and fluorescence data was collected for 10,000 cells. The number of transferred mitochondria was quantified for each cell. (d) MELAS + HEK293T were visualized by DIC and fluorescence microscopy 1 and 2 weeks after mitochondrial transfer. (e) Sanger sequencing of HEK293T, MELAS, and MELAS + HEK293T cells. Arrows denote mtDNA position 3243. (f) Seahorse Extracellular Flux analysis to quantify oxygen consumption rate of HEK293T, MELAS, and MELAS + HEK293T cells. Oligomycin, FCCP, and rotenone/myxothiazol are an ATP synthase inhibitor, uncoupler, and complex I/III inhibitor, respectively. Each data point represents the average of 3 technical replicates and the error bar denotes standard deviation.

Figure 3

Chloramphenicol selection for transferred CAP-R mtDNA retention. (a) Selection of mouse ρ0 or ρ + mutant cells with successfully retained exogenous CAP-R 501-1 mtDNA. (b) Cell lines used with known nuclear and mitochondrial mouse backgrounds. (c) Seahorse Extracellular Flux analysis quantification of basal and maximal cellular respiration in Δmt-ND4, Δmt-ND6, CAP-R 501-1, and L929 ρ0 cells. Two-tailed, unpaired Student’s t-test comparing samples to L929ρ0. * represents significance with * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Black * represents significance for Basal Respiration and Blue * represents significance for Maximal Respiration. The bar height denotes average of 3 replicates and the error bars are the standard deviation. (d) Phosphate buffered saline (PBS) or CAP-R 501-1 mitochondria were transferred into L929 ρ0, Δmt-ND4, and Δmt-ND6 recipient cells and were selected on uridine-deficient, CAP-supplemented media. Four weeks after mitochondrial transfer, colonies were imaged with an inverted microscope and 5 × objective. Scale bar denotes 100 µm. (e) RFLP analysis of CAP-R 501-1, L929 ρ0, L929 ρ0 + CAP-R 501-1, Δmt-ND4 + CAP-R 501-1, and Δmt-ND6 + CAP-R 501-1 bulk culture cells two weeks after mitochondrial transfer. (f) Following Δmt-ND4 + CAP-R 501-1 mitochondrial transfer, cells were cultured in (1) uridine-supplemented media for four days, (2) uridine-deficient, CAP-supplemented media for 24 days, and (3) uridine-supplemented media with or without CAP for 7 days. RFLP analysis of CAP-R 501-1, Δmt-ND4, and Δmt-ND4 + CAP-R 501-1 mitochondria. In (e–f), arrows denote the difference between CAP-S (502 bp) and CAP-R (434 bp) PCR products post-MaeII digestion on a 2.5% agarose gel electrophoresis. CAP-R 501-1 control is the same in each panel. Each of these panels were cropped from different parts of the same gel with the same exposure level.

Figure 4

ρ0 and ρ + mutant recipient cells have restored respiration with transferred CAP-R mitochondria. (a–c) Seahorse Extracellular Flux analysis and quantification of ATP levels contributed by mitochondria (ATPmito) and glycolysis (ATPglyco). Cells were cultured in uridine-deficient, CAP-supplemented media. (a) Analysis of CAP-R 501-1 mitochondrial donor, L929 ρ0 recipient, and L929 ρ0 + CAP-R 501-1 cells. (b) Analysis of CAP-R 501-1 mitochondrial donor, Δmt-ND4, and Δmt-ND4 + CAP-R 501-1 bulk culture cells from three independent transfers. (c) Analysis of Δmt-ND6, CAP-R 501-1, and Δmt-ND6 + CAP-R 501-1 bulk cultures from two independent transfers. (a–c) Two-tailed, unpaired Student’s t-test comparing samples to L929 ρ0, Δmt-ND4, or Δmt-ND6. * represents significance for ATPmito and ‡ represents significance for ATPglyco. * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. ‡ < 0.05, ‡ ‡ < 0.01, ‡ ‡ ‡ < 0.001, ‡ ‡ ‡ ‡ < 0.0001. The bar height denotes average of 4 replicates and the error bars are the standard deviation. (d) Seahorse Extracellular Flux analysis and quantification of ATPmito and ATPglyco in L929 ρ0, Δmt-ND4, CAP-R 501-1, L929 ρ0 + CAP-R 501-1 bulk cultures from three independent transfers, and Δmt-ND4 + CAP-R 501-1 bulk cultures from three independent transfers. Cells were cultured in uridine-supplemented, CAP-supplemented media. Two-tailed, unpaired Student’s t-test comparing samples to Δmt-ND4. * represents significance for ATPmito and ‡ represents significance for ATPglyco. * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. ‡ < 0.05, ‡ ‡ < 0.01, ‡ ‡ ‡ < 0.001, ‡ ‡ ‡ ‡ < 0.0001. There were no statistically significant differences when comparing the samples to L929 ρ0 cells. The bar height denotes average of 5 replicates and the error bars are the standard deviation. (e) Schematic showing summary of ρ + mitochondrial transfer efficiency in a given selection condition.