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Published in final edited form as: Cell Metab. 2016 May 10;23(5):921–929. doi: 10.1016/j.cmet.2016.04.007

Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells

Ting-Hsiang Wu 1,2,14,15, Enrico Sagullo 1,14, Dana Case 1,14, Xin Zheng 1, Yanjing Li 1, Jason S Hong 1, Tara TeSlaa 3, Alexander N Patananan 1, J Michael McCaffery 4, Kayvan Niazi 5,12,13, Daniel Braas 6,7, Carla M Koehler 3,8,9,10, Thomas G Graeber 6,7,9,11,12, Pei-Yu Chiou 2,12,13,*, Michael A Teitell 1,3,9,10,12,13,*
PMCID: PMC5062745  NIHMSID: NIHMS781444  PMID: 27166949

SUMMARY

mtDNA sequence alterations are challenging to generate but desirable for basic studies and potential correction of mtDNA diseases. Here, we report a new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion. The nanoblade rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA. Three stable isogenic nanoblade-rescued clones grown in uridine-free medium showed distinct bioenergetics profiles. Rescue lines 1 and 3 reestablished nucleus-encoded anapleurotic and catapleurotic enzyme gene expression patterns and had metabolite profiles similar to the parent cells from which the ρ0 recipient cells were derived. By contrast, rescue line 2 retained a ρ0 cell metabolic phenotype despite growth in uridine-free selection. The known influence of metabolite levels on cellular processes, including epigenome modifications and gene expression, suggest metabolite profiling can help assess the quality and function of mtDNA modified cells.

Graphical abstract

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INTRODUCTION

Mitochondria are double-membrane eukaryotic organelles of α-proteobacterial origin (Sagan, 1967) that are maternally inherited and help produce energy (ATP) and intermediate metabolites, reducing agents (NADH and FADH2), Fe-S clusters, heme, and steroids. They also generate reactive oxygen species during respiration and regulate apoptosis, Ca2+ homeostasis, and intracellular signaling (McBride et al., 2006). Mitochondria exist in an equilibrium between fused and fragmented morphologies that maintain their shape, size, number, and quality, and they contain their own non-nuclear genome (mtDNA) (Chan, 2012). In humans, the ~16.6 kb circular mtDNA encodes for 13 respiratory chain proteins, 22 tRNAs, and 2 rRNAs. Each nucleated cell contains a few to 100,000 copies of mtDNA that reside in nucleoids (Garcia-Rodriguez, 2007). mtDNA mutations (http://www.mitomap.org) can be silent or cause incurable familial diseases that affect high energy tissues, including brain, heart, and muscle (Taylor and Turnbull, 2005). Cell dysfunction and disease may arise by a critical reduction in mtDNA number or by exceeding a threshold ratio between mutant and wild-type mtDNAs, creating a heteroplasmic state.

Unlike the nuclear genome, strategies for altering mtDNA are limited. Work to overcome the transmission of inherited mtDNA diseases has turned to preimplantation genetic diagnosis to evaluate risk. For women at risk, the transfer of the meiotic spindle-chromosomal complex or a polar body to a donor oocyte, or the transfer of pronuclei to a donor egg, offers the potential for offspring with healthy mtDNA (Richardson et al., 2015). The generation of ‘three-parent’ embryos as an assisted reproduction strategy has generated interest and debate, although these techniques cannot be used for somatic cells or after birth (Richardson et al., 2015; Vogel, 2014).

Alternative strategies for changing the mtDNA content in germ or somatic cells include somatic cell nuclear transfer (Ma et al., 2015; Tachibana et al., 2013) and manipulating the cellular heteroplasmy ratio. Heteroplasmy reduction through a ‘bottleneck’ occurs naturally in early mammalian development and may occur with reprogramming somatic cells to pluripotency (Teslaa and Teitell, 2015). The bottleneck mechanism(s) for reducing heteroplasmy remain unresolved and not all mtDNA haplotypes appear to survive. Mitochondrial targeted nucleases, including restriction endonucleases, zinc finger nucleases, and TALE nucleases, can enrich for specific mtDNAs by incomplete cleavage of target mtDNAs in a mixture, shifting the heteroplasmy ratio (Bacman et al., 2013).

The insertion or replacement of mtDNA sequences by genome editing tools or mitochondrial-targeted adeno-associated viruses (Yu et al., 2012) may also reduce specific mtDNA haplotypes. However, success for these procedures requires DNA repair by non-homologous end joining or homologous recombination, which occur infrequently in mammalian mitochondria (Alexeyev et al., 2013). Therefore, the acquisition of desired mtDNA haplotypes can only be accomplished by transferring mitochondria containing pre-existing mtDNAs into target cells. Successful approaches include cytoplasmic fusion between enucleated mitochondria donor cells and mtDNA eliminated ρ0 cells to generate transmitochondrial cybrid cell lines (Moraes et al., 2001). Also, direct microinjection of isolated mitochondria into somatic cells or oocytes (King and Attardi, 1988; Yang and Koob, 2012) and the transfer of isolated mitochondria, or mitochondrial transfer between cells, in vivo or in co-culture have been reported (Caicedo et al., 2015; Islam et al., 2012; Liu et al., 2014; Spees et al., 2006). However, microinjection is inefficient and it remains unclear whether tunneling nanotube transfer or the ‘spontaneous’ uptake of isolated mitochondria are general phenomena or condition/cell type specific mitochondrial transfer mechanisms.

Recently, we developed a photothermal nanoblade for efficient transfer of small and large objects into mammalian cells by direct cytoplasmic delivery (French et al., 2011; Wu et al., 2011; Wu et al., 2010). Here, we present a proof-of-principle study for nanoblade transfer of isolated mitochondria into ρ0 cells. Metabolomics analyses show the nanoblade is a controlled, reproducible, and general approach for changing the mtDNA haplotype in somatic mammalian cells and may be a potential first step toward reverse mitochondrial genetics.

RESULTS

Photothermal Nanoblade Configuration and Optimization

The apparatus consists of an inverted microscope with a 532 nm nanosecond pulsed-laser that illuminates the field of view through the objective lens. A nanoblade delivery micropipette is mounted on a micromanipulator and connected to an external pressure source (Figures S1A and S1B). The micropipette tip is coated with a light-absorbing titanium thin-film ~100 nm thick (Wu et al., 2011; Wu et al., 2010). This coated tip is positioned to lightly touch the plasma membrane of a cell using the joystick controller. A transient membrane opening is induced by an ultrafast (<300 ns) and localized (<0.4 μm from micropipette rim) cavitation bubble which forms from a laser pulse that rapidly heats the titanium thin film, causing vaporization of adjacent water layers in the culture media (Wu et al., 2011). Bubble expansion and collapse locally punctures the membrane and creates a several micron-long passageway for large cargo delivery that is rapidly repaired (Yamane et al., 2014). Pressure driven fluid flow synchronized to the laser pulse transports cargo into the recipient cell cytosol. The micropipette does not penetrate the cell as in microinjection, is not sealed on the membrane, and membrane disruption is localized to the bubble nucleation site (Wu et al., 2010).

To deliver ~2 μm × 1 μm-sized mitochondria, the nanoblade was fabricated with a 3 μm bore tip inner diameter. This wide orifice prevents clogging and avoids excessive mitochondrial shearing (Figures S2A and S2B). The pulsed laser energy must surpass the superheating threshold for the nanoblade to generate a delivery portal without causing excessive damage and cell death. Optimization of plasma membrane opening efficiency and post-delivery cell viability is cell type specific and established by titration of the laser pulse fluence (mJ/cm2) prior to mitochondria delivery (Wu et al., 2011; Wu et al., 2010). For 143BTK− ρ0 human osteosarcoma cells, a laser fluence of 108 mJ/cm2 yields 64% membrane opening efficiency and 50% viability at 24h (Figure S2C). HeLa cells require 67% more laser energy (180 mJ/cm2) for efficient membrane opening and 80% cell viability. Although photo-irradiation by laser illumination can depolarize mitochondria and cause their clearance by mitophagy (Kim and Lemasters, 2011), minimal to no loss of mitochondrial membrane potential (ΔΨ) occurred in isolated or whole cell mitochondria, respectively, with repeated 108 mJ/cm2 laser pulses (Figure S2D).

Mitochondrial Transfer

Recipient 143BTK− ρ0 cells were labeled with membrane potential insensitive MitoTracker Green and seeded onto a 400 μm × 400 μm square on a patterned glass coverslip to simplify tracking (Figure 1A). HEK293T cells expressing mitochondria-targeted DsRed fluorescent protein (pMitoDsRed) were generated. ~0.5 mg/ml of isolated DsRed mitochondria were loaded into the nanoblade micropipette and delivered into ρ0 cells at ~100 cells/h (Figure 1B). Confocal microscopy with z-stack reconstruction showed donor DsRed and recipient MitoTracker Green-labeled mitochondria intermixed in the cytosol of recipient ρ0 cells 4h after delivery (Figure 1C).

Figure 1. Generating Mitochondrial Rescue Clones by Photothermal Nanoblade.

Figure 1

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(A) Recipient 143BTK− ρ0 cells were seeded on a 400 μm × 400 μm square to facilitate nanoblade delivery, tracking, and clonal selection.

(B) Schematic of mitochondrial delivery by photothermal nanoblade. A 3 μm inner diameter glass microcapillary pipette tip coated externally with titanium is positioned to lightly contact the cell surface. A 532 nm pulsed laser illumination triggers a cavitation bubble to open the membrane with coordinated delivery of donor mitochondria into a cell using a fluid pump.

(C) Representative confocal image of two foci of DsRed labeled donor mitochondria from HEK293T cells in the cytosol of a single 143BTK− ρ0 cell whose endogenous mitochondria are stained with MitoTracker Green (upper left quadrant).

(D) Isolated MDA-MB-453 donor and 143BTK− parent cell mitochondria remain functional and coupled. Mean ± s.d. (n=3).

(E) Two weeks post-nanoblade delivery, donor MDA-MB-453 (and 143BTK− parent, not shown) mitochondria transferred into 143BTK− ρ0 recipient cells generated ‘rescue’ clones that emerged in uridine-free dialyzed media (Left). 143BTK− ρ0 control cells (or 143BTK− ρ0 cells that received 143BTK− ρ0 donor mitochondria, not shown) died and detached from the plate when grown in uridine-free dialyzed media (Right).

MDA-MB-453 human breast carcinoma cells have genomic and mitochondrial DNA sequence polymorphisms and a unique MHC haplotype compared to 143BTK− ρ0 cells, providing distinguishing tags. Oxygen consumption rate (OCR) studies showed isolated mitochondria from 143BTK− parent and MDA-MB-453 cells respire, unlike 143BTK− ρ0 mitochondria (Figure 1D). Electron transport chain (ETC) coupling to oxidative phosphorylation (OXPHOS) expressed as the respiratory control ratio (RCR, state 3/state 4o respiration) provides an estimate of mitochondrial function. MDA-MB-453 and 143BTK− mitochondria had RCRs of 5.1 and 14.7, respectively, whereas 143BTK− ρ0 mitochondria had a negligible RCR.

ρ0 cells are pyrimidine auxotrophs that require uridine supplementation to grow because of an inactive dihydroorotate dehydrogenase (DHOD) enzyme resulting from a non-functional ETC (Gregoire et al., 1984). MDA-MB-453 mitochondria were loaded and nanoblade delivered into ~30 143BTK− ρ0 cells grown in uridine-added medium. Four days post-delivery, cells were shifted to uridine-free medium, with the emergence of respiratory ‘rescue’ clones starting at ~2 weeks (Figure 1E). The frequency of stable, rescue clone generation was 2.1±3.1%, ~10-fold higher than microinjection (Table S1) (King and Attardi, 1988).

Clone Validation and Bioenergetic Analyses

Three nanoblade rescue clones were evaluated. Although ~15% of cells in rescue clones 2 and 3 had PicoGreen mtDNA staining after 2 weeks in uridine-free medium, >85% of cells in all three clones had mtDNA staining after 4 weeks (Figure 2A). Total DNA from donor, parent, ρ0 recipient, and clone 1–3 cells were PCR amplified with primers for mtDNA D-loop hypervariable control region (Figure 2B) and a single nucleotide polymorphism, rs2981582, in the nucleus-encoded FGFR2 gene. Sequencing confirmed all three rescue clones contained exclusively donor mtDNA and ρ0 recipient genomic DNA (Figures 2C and 2D). MHC haplotype analysis showed rescue clone 1 contained the recipient cell nucleus (Figure 2E).

Figure 2. Recipient gDNA – Donor mtDNA Pairing Validates Rescue Clones.

Figure 2

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(A) PicoGreen staining of mtDNA in the cytoplasm of 143BTK− ρ0 cells containing nanoblade-transferred MDA-MB-453 mitochondria at 2 (top row) and 4 weeks (bottom row) post-delivery. 143BTK−ρ0 cells lack mtDNA, do not survive for 4 weeks in uridine− medium, and show only nuclear staining. At 2 weeks in uridine− medium, ~15% of rescue clones 2 and 3 cells have mtDNA, which appear as green puncta in the cytoplasm. By 4 weeks of uridine− selection, >85% of cells in rescue clones 1–3 have mtDNA. Mean ± s.d.

(B) PCR of mtDNA D-loop hypervariable region from rescue clones 1–3 with controls.

(C) Sequencing of mtDNA D-loop hypervariable region revealed multiple single nucleotide polymorphisms (SNPs, red color, arrows) present in rescue clones 1–3 and donor MDA-MB-453.

(D) Rescue clones 1–3 contained the same SNP in the FGFR2 nuclear gene as 143BTK− parent and 143BTK− ρ0 cells (arrow), but a distinct SNP from mitochondrial donor MDA-MB-453 cells.

(E) Human leukocyte antigen (HLA) analysis of rescue 1 shows the same major histocompatibility complex (MHC) loci as 143BTK− parent and 143BTK− ρ0 cells.

Rescue clones 1–3 proliferated at a similar rate to 143BTK− parent cells in uridine-free medium, suggesting the recovery of ETC and DHOD functions (Figure 3A). None of the lines proliferated in 2-deoxyglucose, which blocks glycolysis, but growth in galactose, which favors OXPHOS, was variable (Robinson, 1996), suggesting the clones were not metabolically equivalent. Steady-state ATP levels in clones 1 and 3 were at or above the donor and parent cell levels, respectively, but clone 2 had reduced ATP (Figure 3B). OCR showed basal, maximal respiration, and specific ETC complex I, II, and IV activities for clones 1 and 3 were similar to parent and donor lines, but clone 2 respiration was lower and ρ0 cell respiration absent (Figure 3C). Combined proliferation rate, ATP level, respiratory, and ETC complex activity data suggest rescue clones 1 and 3 are energetically distinct from clone 2, and more similar to the parent cells than to donor or ρ0 recipient cells. The data reveal a range of functional reconstitution by the nanoblade transfer of mitochondria and the restoration of transferred mtDNA.

Figure 3. Bioenergetic Profile of Rescue Clones.

Figure 3

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(A) Proliferation of 143BTK− parent, MDA-MB-453 donor, 143BTK− ρ0, and rescue clone 1–3 cells in the indicated media formulations. Mean ± s.d. (n=3).

(B) Steady-state intracellular ATP levels in arbitrary units. Mean ± s.d. (n=3).

(C) Mitochondria coupling assay (Left) and electron flow assay (Right) as measured by Seahorse XF24 Analyzer. Mean ± s.d. (n=3).

(D) Ratio of citrate synthase enzyme activity to total cellular protein, normalized to 1.0 for the 143BTK− parent line. Mean ± s.d. (n=3).

(E) mtDNA quantification by qPCR, normalized to 1.0 for the 143BTK− parent line. Mean ± s.d. (n=3).

(F) mtDNA-encoded ND1 and ND2 transcript quantification by qRT-PCR, normalized to 1.0 for the 143BTK− parent line. Mean ± s.d. (n=3).

(G) Representative mitochondrial membrane potential quantified by TMRM staining and flow cytometry.

A respiration-independent measure of mitochondrial biomass was provided by citrate synthase enzymatic activity (Zhang et al., 2011), which was similar between rescue clones 1–3, donor, recipient, and parent cells (Figure 3D). ρ0 recipient cells have a granular mitochondrial network (Margineantu et al., 2002) that is retained in rescue clone 1 cells (Figure S3A). Ultrastructure analysis by transmission electron microscopy showed rescue clone 1 mitochondria had tightly stacked cristae with elevated electron density (Figures S3B–S3D). mtDNA levels by qPCR varied >10-fold between rescue clones 1–3 and did not correlate with proliferation rate, ATP level, OCR, or ETC complex activities (Figure 3E). Also, mitochondrial ND1 and ND2 transcript expression did not correlate with mtDNA content for clones 1–3, suggesting differences in mtDNA expression is not the source of variable rescue quality or function (Figure 3F). Finally, ΔΨ was quantified with tetramethylrhodamine methyl ester (TMRM). ρ0 cells hydrolyze ATP in mitochondria to maintain viability and showed a low ΔΨ (Hatefi, 1985). Rescue clones 1 and 3 had fully restored ΔΨ equivalent to parent cells, but rescue clone 2 showed ΔΨ restoration in-between the parent and ρ0 recipient cells (Figure 3G). Thus, ΔΨ provided a potentially superior biomarker for mitochondrial function in rescue clones from nanoblade transfer.

Restoring Metabolism-related Gene Expression

The introduction of mtDNA into ρ0 cells could affect nuclear gene expression directly, by impacting transcription factors, or indirectly, by changing metabolite levels that regulate signal transduction and epigenetics (Kaelin and McKnight, 2013; Teslaa and Teitell, 2015). Changes to nucleus-encoded gene expression patterns by rescuing ρ0 cells through the transfer of isolated mitochondria have not yet been assessed. Therefore, the steady-state expression of 33 genes encoding anapleurotic or catapleurotic enzymes was quantified by qRT-PCR (Figure S4A &B). An unbiased, systems level assessment of steady-state gene expression was obtained by principle component analysis (PCA), which investigated relationships amongst all 6 cell lines (Figure 4A). Rescue clones 1 and 3 had gene expression profiles similar to the parent, whereas rescue clone 2 was similar to ρ0 recipient. Donor cells showed a distinct gene expression profile compared to the other cell lines. The data indicate nanoblade transfer of mtDNA resets the nucleus-encoded metabolic enzyme gene expression pattern partially (clone 2) or almost completely (clones 1 and 3) to the parental and not the donor cell nucleus in an isogenic nuclear background.

Figure 4. Metabolic Gene Expression and Metabolite Profile of Rescue Clones.

Figure 4

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(A) Expression of 33 genes involved in TCA cycle metabolism quantified by qRT-PCR and normalized to the ribosomal 36B4 gene (see Figure S4B). PCA shows the grouped relationships between the six cell lines studied.

(B) Relative levels of twelve TCA cycle proximate metabolites quantified by LC-MS/MS and normalized to the 143BTK− parental line. Mean ± s.d. (n=3).

(C) Heatmap of 96 metabolites measured with an ANOVA p-value equal to or less than 0.05. Samples were clustered using a Pearson correlation matrix.

(D) PCA using the fractional contribution from U-13C glucose to all measured metabolites.

(E) PCA using the fractional contribution from U-13C glutamine to all measured metabolites.

Metabolite Profiling

Intracellular metabolite levels have not been quantified for mtDNA modified systems. A liquid chromatography-mass spectrometry (LC/MS)-based metabolomics assay assessed TCA cycle-related and select cytosolic metabolites that could impact gene expression and cellular biosynthetic functions. Of 12 TCA cycle related metabolites (sans pyruvate, whose quantification was highly variable), alpha-ketoglutarate (α-KG) and citrate (Cit) stood out as strongly depleted in ρ0 recipient cells compared to parent cells (Figure 4B). All 3 rescue clones showed increased α-KG and citrate above those in the ρ0 recipient. In contrast, ρ0 recipient cells accumulated the oncometabolite 2-hydroxyglutarate (2-HG) and succinate (Succ) relative to parent cells. All 3 rescue clones resolved the block at succinate dehydrogenase (ETC complex II), although clone 2 retained elevated 2-HG levels in contrast to clones 1 and 3. An unbiased assessment of 96 metabolites, including amino acids and nucleotides, was obtained by hierarchical clustering (Figure 4C) and PCA (Figure S4C), to evaluate the systems level rescue of ρ0 recipient cells by nanoblade. Rescue clones 1 and 3 clustered with parent cells whereas rescue clone 2 grouped together with ρ0 recipient cells, and donor cells stood apart from both of these clusters. Similar to nucleus-encoded gene expression, nanoblade transfer of mtDNA restores the steady-state metabolite pattern of ρ0 recipient cells partially (clone 2) or almost completely (clones 1 and 3) to the parent and not the donor cells in an isogenic nuclear system.

The activities of metabolic pathways and the contributions of nutrients to specific intracellular metabolites were examined with stable isotope labeling. Fully labeled [U-13C] glucose (Glc) and glutamine (Gln) provided the fractional contribution to network metabolites for recipient, parent, donor, and rescue clone 1–3 lines (Figures S4D). Rescue clones 1 and 3 grouped with the parent line by PCA, while rescue clone 2 was metabolically more similar to ρ0 recipient cells and the donor line was metabolically distinct from all other analyzed cell lines. Overall, the data suggested gene expression, metabolic network activity, and metabolite levels in ρ0 recipient cells were almost completely restored to the parent cell levels in rescue clones 1 and 3, but not in rescue clone 2.

DISCUSSION

In a proof-of-principle study, we demonstrated the photothermal nanoblade can transfer isolated mitochondria from an allogeneic cell type to restore metabolic gene expression, global energetic, and metabolite profiles. The nanoblade enables comparisons of different mitochondria and their metabolic performance in an isogenic nuclear background. The mtDNA level, mtRNA expression, mitochondrial biomass, and ultrastructure did not correlate with rescue clone performance. A broad range of rescue clone respiration and respiratory capacity unrelated to mtDNA or mtRNA levels was also reported for clones previously generated via microinjection (King and Attardi, 1989) and cybrid fusion (Chomyn et al., 1994). Instead, system wide activities as reflected by ΔΨ and global metabolite recovery were a better predictor of rescue clone quality and function. Hierarchical clustering and PCA of steady-state metabolite levels and the fractional contribution of glucose and glutamine to isotopologues indicate rescue clones 1 and 3 are restored to the parental metabolic profile. Interestingly, the partially rescued clone 2 survived uridine-free selection and still remained most similar to ρ0 cells, possibly by sufficient recovery of ETC function to increase DHOD activity and uridine production. This idea is supported from the reversal of the ETC complex II (succinate dehydrogenase) block and full recovery of parent succinate levels for all 3 rescue clones. All 3 rescue clones adopted features of the recipient and not the donor.

Compared to cell fusion, the photothermal nanoblade can deliver washed, isolated mitochondria, minimizing the transfer of other cytosolic biomolecules that could impact biological functions in cells, such as microRNAs, metabolites, and signaling molecules. Initial applications for the nanoblade approach include more detailed studies of mtDNA expansion kinetics with or without manipulations of regulators of mtDNA replication or mitochondrial fusion/fission. A defined ratio of mixed donor mitochondria can also be simultaneously transferred into a cell, which aid in studies of heteroplasmy mtDNA competition, nuclear/mitochondrial genome compatibility, and kinetics of metabolic rewiring. Finally, the nanoblade could dissect mechanisms of inefficient, uncontrolled, and spontaneous cellular uptake of mitochondria without cell fusion (Katrangi et al., 2007; Kitani et al., 2014; Masuzawa et al., 2013).

Mitochondria transfer by photothermal nanoblade is ~2% efficient, which is higher than cell fusion (0.0001–0.5%) (Table S2 and Table S3). However, the nanoblade is low throughput because mitochondria are transferred into successive individual cells. Due to variable recovery of recipient cell function seen with all reported mitochondrial transfer approaches, a larger number of clones in each experiment are needed to obtain a spectrum of clone performance and enable the selection of optimal clones for a particular purpose. A potential solution is the recent development of a biophotonic laser-assisted surgery tool (BLAST) for the massively parallel transfer of large cargo into mammalian cells using the same biophysical principle as the one-cell-at-a-time nanoblade, with appropriate modifications and optimization for mitochondrial transfer reactions (Wu et al., 2015).

EXPERIMENTAL PROCEDURES

Standard procedures were followed for cell culture, sequencing, oxygen consumption studies, ATP quantification, microscopy, citrate synthase and ΔΨ assays, qPCR and qRT-PCR, and metabolomics, as described in the supplemental procedures.

Photothermal Nanoblade

Borosilicate glass micropipettes ~3 μm tip in diameter and ~5 mm long were generated using a micropipette puller (Sutter Instrument, P-97). A ~100 nm titanium thin film was deposited on the tip and outer walls of pulled micropipettes using a magnetron sputterer deposition system (Denton Vacuum, Discovery 550). Photothermal nanoblade operation and optimization is described in the supplemental procedures.

Mitochondrial Isolation and Delivery

Mitochondria isolation was performed using standard methods (Frezza et al., 2007). Briefly, MDA-MB-453 cells were detached from an 80–90% confluent 15 cm petri dish using a cell scraper and suspended in 1 ml ice-cold isolation buffer, IBc (10 mM Tris/MOPS, 5 mM EGTA/Tris and 0.2 M sucrose, 1 mM protease inhibitor (Sigma, P8340) and 0.2% BSA, adjusted to pH 7.4). Cell suspension was homogenized using a Teflon pestle in a glass potter with 30 strokes on ice and the mitochondrial fraction obtained after centrifugation washes. Isolated mitochondria pellet was resuspended in ice-cold experimental buffer, EBc (125 mM KCl, 10 mM Tris/MOPS, 1 mM EGTA/Tris, 0.1 M sucrose, 1 mM KH2PO4, 1 mM protease inhibitor and 0.2% BSA, adjusted to pH 7.4). Resuspended mitochondria (0.5 mg/ml protein concentration) were kept on ice until delivery. ~8 μl of isolated mitochondria suspension was loaded into a nanoblade micropipette. Nanoblade deliveries were done into ~30 cells per experiment (Table S1) and successful delivery was visualized by transient cytosol volume expansion.

Statistical Analysis

Experiments were carried out in triplicate and data represents the mean ± standard deviation. A Student t-test was used to determine p values.

Supplementary Material

1
2

Highlights.

  • Proof-of-principle photothermal nanoblade transfer of mitochondria is reported.

  • Transfer into 143BTK− ρ0 cells generated rescue clones with recovered respiration.

  • Mitochondrial transfer reset metabolic enzyme gene expression patterns.

  • Two of 3 rescue clones showed metabolite profiles similar to 143BTK− parent cells.

Acknowledgments

Supported by UC Discovery Biotechnology grant 178517, Air Force Office of Scientific Research grant FA9550-15-1-0406, NIH grants GM007185, GM114188, GM073981, GM061721, EB014456, CA009056, CA90571, CA156674, CA185189, and CA168585, NSF grant CBET-1404080, CIRM grants RB1-01397 and RT3-07678, Prostate Cancer Foundation Challenge Award, Broad Stem Cell Research Center Training Grant and Innovator Award, American Cancer Society Research Scholar Award RSG-12-257-01-TBE, Melanoma Research Alliance Established Investigator Award 20120279, National Center for Advancing Translational Sciences UCLA CTSI Grant UL1TR000124, and by NanoCav, LLC. The authors thank S. Rabizadeh (Nantworks, LLC) for helpful discussions and support.

Footnotes

AUTHOR CONTRIBUTIONS

T.-H.W. and E.S. engineered the photothermal nanoblade platform. T.-H.W., E.S., D.C., X.Z., J.S.H., A.N.P., J.M.M., and K.N. generated mitochondrial rescue cell lines and molecular and cellular data. Y.L., T.T., D.B., and T.G.G. generated and analyzed metabolite profiling data. T.-H.W., C.M.K., P.-Y.C., and M.A.T. designed the research and/or analyzed data with help from all other authors. T.-H.W. and M.A.T. wrote the paper with help from E.S., D.C., D.B., C.M.K., and P.-Y.C.

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Supplementary Materials

1
NIHMS781444-supplement-1.pdf (1.1MB, pdf)
2
NIHMS781444-supplement-2.xlsx (15.5KB, xlsx)

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