Generation of constructs

The protein sequence of AflIII restriction enzyme (protein ID: ADO24177.1) without the N-terminal Methionine was back translated and codon optimized for translation in Drosophila in Gene Designer 2.0 (https://www.dna20.com/resources/genedesigner). The N-terminal tag directing protein import into the mitochondrial matrix (60 amino acids from the N terminus of Drosophila Aconitase (CG9244), plus a C-terminal Alanine) was identified with the Target P 1.1 program (http://www.cbs.dtu.dk/services/TargetP/). It was then appended to the N terminus of AflIII, generating mitoAflIII. To build the pUASt-mitoAflIII, a mitoAflIII fragment was gene synthesized by DNA2.0 and cloned into pWALIUM 20 (https://plasmid.med.harvard.edu/PLASMID/GetVectorDetail.do?vectorid=500) between XbaI and NdeI sites.

A mitochondria-targeted version of T4 DNA ligase was built as follows. The coding sequence (CDS) of T4 DNA ligase was PCR amplified from Escherichia coli carrying T4 bacteriophage. An out of frame synthetic intron was inserted into the CDS of T4 ligase to facilitate bacterial cloning and manipulation. To generate pUASt-mitoT4ligase, the N-terminal mitochondrial targeting sequence utilized with AflIII, T4 ligase and two translations enhancers IVS (a small 5′-untranslated region (UTR) intron from the Drosophila Mhc gene, CG17927) and p10 3′-UTR (the terminator sequence from the AcNPV p10 gene) were cloned into pWALIUM 20 cut at BglII and HpaI sites). To build constructs for overexpression of parkin, drp1 and Atg8a, a mitoT4ligase fragment in pUASt-mitoT4ligase plasmid was replaced with corresponding CDSs. CDSs of parkin, drp1, Atg8a and ATPIF1 were PCR amplified from Drosophila cDNA, reverse-transcribed with an oligo-dT primer from total RNA.

A three-transgene construct (pIFM-mitoAflIII, pIFM-mitoT4lig and pIFM-Gal4) that directs IFM-specific expression of mitochondrially targeted AflIII and T4 ligase, and the yeast transcriptional activator Gal4, was built in pWALIUM 20 (Fig. 1c; GenBank, KX696451). The Flightin promoter (pIFM) was PCR amplified from Drosophila melanogaster genomic DNA, using primers 5′-CGTTCCCGTGATAGAGTAACGGTTCCT-3′ and 5′-CAGCTAAAACTAGGACATTGGGTCCACTG-3′, 543 bases 5′ to the start site of transcript variant B of the Flightin gene (CG7445) and 23 bases 5′ of its 5′-UTR, respectively. The fragment between the two gypsy insulators in pWALIUM 20 was removed with PstI and HpaI. pIFM, IVS, mitoAflIII and p10 3′-UTR fragments were then ligated together. The resulting plasmid was then cut with StuI and pIFM-mitoT4lig was cloned, 5′–3′, from the gypsy insulator towards the attP site. Finally, pIFM-Gal4 was cloned, 5′–3′, into the XhoI site, from the White gene towards the attP site (Fig. 1c).

Transfection of Drosophila S2 cells

Drosophila S2 cells were maintained using standard protocols (Invitrogen, #R690-07). Transient transfections were performed using FuGene HF (Roche) with the following plasmids: pUASt-mitoAflIII, pUASt-mitoT4lig, pMT-Gal4 and pJFRC81 and pCaSpeR-hs. pJFRC81 (pUASt-eGFP-p10) was added to visualize the efficiency of transfection and activation; and pCaSpeR-hs (empty pHsp70 vector) to control for the amount of transfected DNA. pMT was induced with 400 μM of CuSO 4 7 h after transfection. Thirty hours post transfection, DNA was extracted from S2 cells and analysed by PCR.

PCR detection of the engineered mtDNA deletion

DNA from S2 cells and Drosophila tissues was extracted with the DNeasy Blood and Tissue kit (Qiagen). The following two primers flanking the two AflIII sites in Drosophila mtDNA were used to PCR mtDNAΔ: 5′-ATCATATTTGTCGAGACGTTAATTATGGTTG-3′ and 5′-GAAATGAAATGTTATTCGTTTTTAAAGGTATCTAG-3′. These primers amplify either a 481 bp product from mtDNAΔ molecules or a 3,065 bp product from mtDNAWT molecules. The following primers complementary to the mt:CytB locus, located inside the deleted fragment, were used to amplify a 498 bp product present in mtDNAWT: 5′-GGACGAGGAATTTATTACGGTTCATA-3′ and 5′-GTGTTACTAAAGGATTTGCTGGAAT-3′. LongAmpTM Taq DNA polymerase (New England BioLabs, NEB), with a 65 °C elongation temperature, was used for PCR with the following conditions: (94 °C for 20 s, 51.5 °C for 13 s, and 65 °C for 25 s) × 32 cycles. PCR amplicons were sequenced directly. If sequencing showed the presence of different templates (as determined by ambiguity at or near the post-cleavage AflIII site), they were first cloned into TOPO TA vector (Invitrogen) and then multiple individual clones were sequenced.

Drosophila strains and genetics

Flies were maintained on the standard cornmeal/soy flour/yeast fly food at 25 °C with a 12H/12H light and dark cycle. The following fly stocks were obtained for this study: da-Gal4 (Bloomington #8641); elav-Gal4 (Bloomington #8765); Mef2-Gal4 (Bloomington #27390); ey-Gal4 (Bloomington #5534 & #5535); r4-Gal4 (Bloomington #33832); pUASt-mitoGFP58; pUASp-mCherry-Atg8a (Bloomington # 37750, from which Dr1/TM3, Ser1 was removed); pUASt-Atg1(6B) III (gift from Thomas Neufeld, University of Minnesota); Atg1 RNAi line (Bloomington #26731); Atg8a RNAi line (Vienna Drosophila RNAi Center #109654); Atg8aKG07569/FM7c (Bloomington #14639); pUASt-Pink1 (ref. 38); pUASt-RNAi-Marf58.

Constructs were injected at Rainbow Transgenic Flies, Inc. (http://www.rainbowgene.com). The constructs containing pIFM-Gal4 were inserted at the P{CaryP}attP1 site on the second chromosome (Bloomington #8621), while constructs with trans-activated genes were inserted at the P{CaryP}attP2 on the third chromosome (Bloomington #8622). Transgenic flies were balanced against the same background: w1118; CyO/snaSco and w1118; TM3, Sb1/TM6B, Tb1, respectively.

MitoGFP and pUASp-mCherry-Atg8a expression and visualization

To visualize mitochondria in the Drosophila IFMs pUASt-mitoGFP58 was recombined into two genetic backgrounds: P{pIFM-mitoAflIII, pIFM-mitoT4lig, pIFM-Gal4}attP1 and P{pIFM-Gal4}attP1. The two generated lines were crossed to flies carrying the marker of autophagy, pUASp-mCherry-Atg8a46. The IFMs were dissected from 3-day-old male flies and fixed for 35 min in PBS with 4% paraformaldehyde and 0.2% Triton X-100. After washing three times in PBS and 0.2% Triton X-100, samples were mounted into Vectashield mounting medium and imaged on a confocal scope (Olympus FluoView-1000). Images were acquired with a × 60 (numerical aperture=1.30) objective and 1,024 × 1,024 resolution at 12 bits per pixel. Each channel (GFP and mCherry) was scanned consecutively at 12.5 μs per pixel. Three slides were prepared and examined for each genetic group. Six images applying a twofold zoom were acquired for quantification (Fig. 4b). A higher magnification (a fourfold zoom) was used to acquire images for presentation (Fig. 4a).

Quantification of mtDNAΔ with qPCR

Aged male flies were fixed in 100% ethanol and stored at 4 °C. After fixing flies for at least 14 h, IFMs were dissected in 100% ethanol. Twenty male flies were used per replicate sample. Ethanol was then removed and the sample dried on a heatblock for 15 min at 90 °C. DNA was extracted from dried IFMs with the NucleoSpin Tissue XS kit (Macherey-Nagel). DNA concentration was measured with the NanoDrop ND-1000 (Thermo Scientific) and adjusted to 10 ng μl−1.

The abundance of mtDNAΔ was quantified relative to both mtDNAtotal and nucDNA using real-time qPCR. The relative quantification of mtDNA deletions with qPCR has previously shown to correlate well with estimates based on Southern blotting70. Real-time qPCR was performed with the SYBR Green I Master kit (Roche) on the LightCycler 480 real-time PCR system (Roche). An elongation step of PCR was done at 65 °C for 25 s. Amplifications of four loci were assessed with qPCR: mt:cytB (specific for mtDNAWT), mt:AflIIIΔ (specific for mtDNAΔ), mt:NADH5 (mtDNAtotal) and nuc:Tube (nucDNA single-copy locus; CG10520; Supplementary Table 4; Fig. 2a). The first two loci assessed the amounts of mtDNAWT and mtDNAΔ, respectively; the second two loci provided different references for normalization of the amount of mtDNAΔ (ΔCt). Melting curve analysis did not identify any primer-dimer peaks in melting profiles. To estimate the dynamic range, efficiency and sensitivity for each primer set, we constructed a standard curve covering three orders of magnitude of total DNA concentration. DNA was extracted from the dissected IFMs of w1118; P{pIFM-mitoAflIII, pIFM-mitoT4lig, pIFM-GAL4}attP1/CyO 10-day-old male flies. The four primer sets have identical efficiencies and sensitivity though the entire range (Fig. 2b). Based on this, we applied a simple ΔΔCt quantification algorithm to measure the effect of a tested gene (OE or KD) on the level of mtDNAΔ. The w1118; P{pIFM-mitoAflIII, pIFM-mitoT4lig, pIFM-GAL4}attP1/+ 10-day-old male flies were the reference sample for all comparisons. At least four biological replicates were analysed for each genetic background. The calculations of ΔΔCt, fold difference and normalized percentages were performed in Microsoft Excel for Mac 2011. Equations of the ΔΔCt algorithm are presented in Supplementary Methods.

Toluidine blue staining and transmission electron microscopy

Thoraces from 10-day-old P{pIFM-Gal4}attP1 (wild type) and P{pIFM-Gal4, pIFM-mitoAflIII, pIFM-mitoT4lig}attP1 (mitoAflIII flies) male flies were dissected, fixed in paraformaldehyde/glutaraldehyde, post-fixed in osmium tetraoxide, dehydrated in ethanol and embedded in Epon. Blocks were cut to generate 1.5-μm-thick sections using a glass knife, or 80-nm-thick sections using a diamond knife on a microtome (Leica, Germany). Toluidine blue was used to stain 1.5-μm-thick tissue sections. Thin sections (80-nm thick) were stained with uranyl acetate and lead citrate, and examined using a JEOL 100C transmission electron microscope (UCLA Brain Research Institute Electron Microscopy Facility). At least six thoraces were examined for each genotype.

Flight assay

Flight performances of mitoAflIII and wild-type 10-day-old male flies were compared using the ‘cylinder drop assay’43. Five groups of 30–50 flies of each type were introduced into the top of a 500-ml graduated cylinder whose internal walls were coated with paraffin oil. Wild-type flies quickly initiate horizontal flight, striking the wall close to the entry level, whereas poor fliers land at lower levels or at the bottom of the cylinder. The distribution of heights at which the flies stuck in the oil reflects their flying performance.

Quantification of transcript abundance with RT–qPCR

The w1118; P{pIFM-mitoAflIII, pIFM-mitoT4lig, pIFM-GAL4}attP1/CyO (mitoAflII) transgenic male flies were used to quantify the transcription of pIFM-mitoAflIII. Five time points were used to probe the mRNA abundance of mitoAflIII: 1, 3, 5, 7 and 9 days post eclosion. The expression of Atg1 and Atg8a gene was analysed in the same transgenic flies at day 3 post eclosion. Total RNA was extracted from the dissected thoraces and legs of flies using the mirVana miRNA isolation Kit (Ambion). To remove DNA contamination, 3 μg of total RNA was treated with the Turbo DNA-free kit (Ambion). Then, cDNA was synthesized from 500 ng of RNA with SuperScript III reverse transcriptase (Invitrogen) using an oligo-dT primer. qPCR with reverse transcription (RT–qPCR) was performed with the SYBR Green I Master kit (Roche) and the CFX96 Real-Time PCR detection system utilizing the Bio-Rad C1000.

Primers used for RT–qPCR are listed in Supplementary Table 5. We placed one primer directly on a splice junction to avoid amplification of genomic DNA. A melting curve analysis was performed to confirm the absence of any primer-dimer peak in the melting profile. Three dilution series (1/1, 1/10 and 1/100) were used to build a standard curve and estimate the dynamic range, efficiency and sensitivity for each primer set. The mRNA abundances of mitoAflIII, Atg1 and Atg8a transcripts were normalized to β glucuronidase (βGlu) mRNA, according to the ΔCt method with an efficiency correction (Supplementary Methods). Four biological replicates were analysed per sample. mRNA abundances of mitoAflIII at different time points were compared with the value at 1 day post eclosion, which was given the value of 100% (Fig. 1e). Three-day-old w1118; P{pIFM-GAL4}attP1/CyO male flies served as a reference for estimation of changes in Atg1 and Atg8a mRNA abundance in the mitoAflII flies.

Target-primed tpRCA and immunochemistry

We performed target-primed RCA in whole-mount IFMs dissected from male flies using a modified version of a published method42. In brief, mtDNA in fixed tissue was cleaved with the restriction enzyme EcoRI and then made single stranded using λ 5′–3′ exonuclease. Two different padlock probes were then hybridized to mtDNA. One hybridization site was located near (44 bp) an EcoRI site present in widl type and mutant mtDNA (green oval in Fig. 3a). A second probe hybridizes near (66 bp) an EcoRI site that is brought close to the remaining AflIII site following mitoAflIII cleavage and re-ligation, but located 1.71 kb away from the probe binding site in widl-type mtDNA (red oval in Fig. 3a). After ligation of the padlock probes, rolling-circle amplification using Φ29 DNA polymerase was carried out, using the nearby 3′-end provided by EcoRI cleavage as a primer. This results in many rounds of amplification of the padlock probe sequence, which is detected through hybridization with fluorescently labelled probes (dpMtDNAtotal-AlexaFluor488; and dpMtDNAΔ-TAMRA; green and red loci, respectively; Fig. 3). mtDNAΔ can be specifically identified using this approach because the ability of a 3′-end in fixed tissue to act as a primer drops markedly with distance from the padlock probe42, and thus only occurs when one of the EcoRI sites has been brought near the post-cleavage AflIII site (Fig. 3a, details below). We note that the efficiency of tpRCA-based labelling of nucleoids in muscle is low, as is also seen in mammalian cells42. Because of this, and the fact labelling efficiency can vary several fold, tpRC is not used to estimate the absolute levels of mtDNA. It can only be used to determine the fraction that is mtDNAΔ.

All reactions were performed inside 0.2 ml PCR tubes. At each step, we incubated samples with an enzyme or probe at 4 °C for 1 h, to allow for tissue penetration, before raising the temperature to 37 °C. The IFMs were dissected from 10-day-old flies and fixed in 4% formaldehyde in PBS with 0.3% Triton X-100 for 35 min. The fixed tissue was rinsed three times for 5 min in wash A (PBS with 0.2% Triton X-100) at room temperature (RT), heated at 75 °C for 15 min and chilled on ice for 2 min. DNA was digested using 0.5 U μl−1 of EcoRI HF (NEB) at 37 °C for 40 min, and rinsed three times in wash A at RT for 5 min. Hybridization target sequences for two padlock probes (Supplementary Table 6) were located either near an EcoRI site present in both mtDNAWT and mtDNAΔ (mtDNAtotal, green oval; Fig. 3a), or near an EcoRI site only brought near the hybridization site through creation of a deletion (mtDNAΔ, red oval; Fig. 3a). We applied 0.2 U μl−1 of the 5′–3′ λ exonuclease (NEB) at 37 °C for 15 min to make a single-stranded DNA target for complementary padlock probes.

Two 5′-end phosphorylated padlock probes served as a template for target-primed RCA: ppMtDNAtotal and ppMtDNAΔ. The padlock probe hybridization site for ppMtDNAtotal is located 44 bp upstream of one EcoRI site, while the padlock probe hybridization site for ppMtDNAΔ is located 1,752 bp upstream of a second EcoRI site in mtDNAWT, and 66 bp upstream of an EcoRI site in mtDNAΔ (Fig. 3a). After three rinses in wash A at RT for 5 min, we hybridized 185 nM of both padlock probes under the published conditions42 at 37 °C for 40 min. To remove unbound padlock probes we rinsed sample in wash B (2 × SSC, 0.02% Triton X-100) at 37 °C for 5 min, and then in wash A three times each for 5 min at RT. Padlock probes were circularized using 0.1 U μl−1 T4 DNA ligase (NEB) in the supplied buffer supplemented with 500 μM of ATP (NEB) at 4 °C overnight. Samples were then rinsed in wash B at 37 °C for 5 min and three times in wash A at RT for 5 min. We performed the RCA reactions using 1 U μl−1 of Φ29 DNA polymerase (NEB) in the supplied buffer supplemented with 5% glycerol and 200 μg μl−1 of BSA at 37 °C for 40 min. Samples were rinsed in wash A three times at RT for 5 min, and then post-fixed in 4% formaldehyde in PBS with 0.3% Triton X-100 for 20 min each. After three rinses in wash A, we hybridized 250 nM of fluorophore-tagged detection probes (Supplementary Table 6) under the same conditions as for the padlock probe hybridization, at 37 °C for 90 min. The samples were rinsed three times in wash A at RT for 5 min before mounting them in VectaShield (Vector Laboratories) on a microscope slide.

The samples used for immunostaining were blocked in 5% BSA in wash A at RT for 1 h. After blocking, samples were rinsed three times in wash A at RT for 5 min, and stained with primary and secondary antibodies overnight at 4 °C. After primary and secondary staining, samples were extensively rinsed, four times in wash A at RT for 20 min each. Mitochondria were immunostained using mouse monoclonal (15H4C4) anti-ATP5A (Abcam #14748) at a 1/300 dilution and goat anti-mouse Alexa Flour 405 (Abcam #175661) at a 1/250 dilution.

Imaging of tpRCA

Images of in situ tpRCA were acquired with the Olympus FV-1000 confocal microscope using a × 60 (numerical aperture=1.30) objective and 1,024 × 1,024 resolution at 12 bits per pixel. Each channel was scanned consecutively at 4 μs per pixel applying a threefold zoom. For each genotype, tissue from six flies was examined, with four or more images per fly, and a single 400 × 400 image is displayed in Figs 5b and 6b.

Image quantification

ImageJ 2.0 was used for image analysis and quantification. For each colour channel, a threshold was determined from a channel’s histogram and applied to remove background pixels. To quantify numbers of fluorescent loci (mCherry-Atg8a, Alexa Flour 488 and TAMRA), colour channels were split, converted to 8 Bit grey scale and then to binary images. Numbers of fluorescent foci were counted with a particle analysis tool in ImageJ. Total numbers of fluorescent foci counted were similar for all genotypes and samples.

Quantification of mtDNAΔ using tpRCA

In order for a padlock probe to be able to hybridize to its target, and to be used as a substrate for rolling-circle amplification, several things must happen. First, the 5′–3′ exonuclease must degrade one strand of mtDNA, beginning at the cleaved EcoRI site, to provide a probe hybridization site. Second, following hybridization of the padlock probe, Φ29 DNA polymerase must shorten the unprimed 3′-end so that it becomes located sufficiently near the probe hybridization site to function as a primer. Both of these exonucleolytic events are likely to be inhibited in a distance-dependent manner in an in situ preparation of formaldehyde fixed tissue, since in vivo mtDNA is bound to varying degrees, depending on context, by mtDNA-packaging proteins such as TFAM1. Larsson et al.42 observed such distance-dependent behaviour, with padlock probes located close to the restriction enzyme-created end being detected twice as frequently as those located 134 bp away. In our experiments, the mtDNAΔ padlock probe is located 1.7 kb away from the exposed 3′-end in wild-type mtDNA, but only 66 bp away in mtDNAΔ. Thus, failure of one or both of the above exonucleases to process the intervening mtDNA provides a mechanistic basis for our ability to specifically detect mtDNAΔ. With respect to our ability to quantify the fraction of mtDNAtotal represented by mtDNAΔ using tpRCA, we note that target sites for mtDNAΔ and mtDNAtotal padlock probes are located at different distances from their closest EcoRI cut sites: 66 and 44 bases, respectively. Assuming that the ability of a 3′-end to prime tpRCA is a linear function of distance (based on the above proposed mechanisms, and the observations of Larsson et al.)42, we applied a correction factor for different efficiencies of RCA of the circularized padlock probes by adjusting the percentage of mtDNAΔ (red) to mtDNAtotal (green) loci on tpRCA images by 3/2.

mitoAflIII fly feeding experiments

The P{pIFM-mitoAflIII, pIFM-mitoT4lig, pIFM-Gal4I}attP1/CyO male flies were used for feeding experiments. All flies were kept under the same conditions at 25 °C with a 12H/12H light and dark cycle. Twenty flies were introduced into a vial, and flies were transferred into a new vial every other day. Rapamicyn (99+%, Alfa Aesar) was dissolved in 100% ethanol to a 40 mM stock concentration. It was gradually diluted in water to a 200 μM working concentration. Drosophila food was prepared from Instant formula 4–24 (Carolina) dissolved in water with 200 μM rapamycin. Diluted ethanol alone was added to the control food. Newly enclosed flies were kept for 10 days on 200 μM of rapamycin.

Quantification of mtDNAΔ from mitoAflIII flies fed on supplemented diets

Total DNA was extracted from dissected IFMs of 10-day-old male flies. The same qPCR primers (Supplementary Table 4) and ΔΔCt quantification algorithm were used to estimate changes in levels of mtDNAtotal and mtDNAΔ molecules between the control flies and flies fed on food supplemented with rapamycin. Because each group (experimental and control) was fed on Instant formula 4–24 (Carolina), we could not normalize the fold change in mtDNAΔ induced by rapamycin to the level of mtDNAΔ in the mitoAflII flies fed on the standard fly food. Instead, changes in abundance of the mtDNAtotal and mtDNAΔ are presented as percentages relative to the respective estimates in the control flies, which were given the value of 100%. Four biological replicates were analysed for each feeding experiment.

Statistical analysis

Statistical analysis was performed in JMP 8.0.2 by SAS Institute Inc. Observations from each genetic background were compared with corresponding values from mitoAflIII flies (Figs 5a and 6a). P values were calculated for a two-sample Student’s t-test with unequal variance.

Data availability

The sequence of plasmid used to generate mitoAflIII flies is deposited in GenBank under accession code KX696451.