Topological transformation of mitochondria under serum starvation

To investigate the relationship between mitochondrial morphology and stress adaptation under starvation, we tracked mitochondria by fluorescence microscopy using mitochondrial matrix targeted GFP (mtGFP) under serum starvation. The increased appearance of donut and swollen mitochondria under starvation was obvious (Figure 1A). Time course images showed that these 2 morphologies arose in different ways: swollen mitochondria were formed by shortening and rounding of tubular mitochondria, while donuts arose when tubular mitochondria bent, formed a ‘C’ shape, and self-fused (Figure 1B). To distinguish swollen mitochondria from fragmented mitochondria, we defined swollen mitochondria as those with a diameter of at least twice the typical width of a tubule. As we previously showed that short-term mitochondrial uncoupler treatment induces robust donut formation [28], we also performed carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) treatment as a control. We found that both donut and swollen mitochondria increased under serum starvation while predominantly donuts were formed upon 30 min FCCP treatment (Figure 1C). The number of donut and swollen mitochondria progressively increased during prolonged starvation time from 0 h to 36 h (Figure 1D), while cell viability was not affected (Figure S1).

Topology-dependent, bifurcated mitochondrial quality control under starvation

All authors

Yanshuang Zhou, Qi Long, Hao Wu, Wei Li, Juntao Qi, Yi Wu, Ge Xiang, Haite Tang, Liang Yang, Keshi Chen, Linpeng Li, Feixiang Bao, Heying Li, Yaofeng Wang, Min Li & Xingguo Liu

https://doi.org/10.1080/15548627.2019.1634944

Published online:

04 July 2019

Figure 1. Topological transformation of mitochondria under serum starvation. (A) Representative images of mitochondrial morphology under serum starvation and FCCP treatment. U2OS cells expressing mtGFP were starved for 24 h or treated with FCCP for 0.5 h. Scale bar: 5 μm. (B) Time series of the formation of swollen and donut mitochondrion under serum starvation in U2OS cells (swollen mitochondrion, swell; donut mitochondrion, donut). Scale bar: 1 μm. (C) Quantification of swollen and donut mitochondria per cell in U2OS cells under 24-h serum starvation or 0.5-h FCCP treatment (150–200 cells from 3 independent experiments). (D) Morphometric analysis of mitochondria in U2OS cells under serum starvation for the indicated time (150–200 cells from 3 independent experiments). (E) Representative TEM images of donut and swollen mitochondria in U2OS cells after 1-h FCCP treatment. Scale bar: 1 μm. The white arrowheads indicate mitochondrial cristae. (F) The ratio of the sum of cristae length to the mitochondrial area from TEM images (at least 40 mitochondria in each group). (G) Time series of the fate of swollen and donut mitochondrion under starvation in U2OS cells. Scale bar: 1 μm. (H) The percentage of swollen and donut mitochondria reverting to tubular mitochondria under 24-h starvation in U2OS cells (>100 cells from 3 independent experiments). Data in (C), (F) and (H) are displayed as mean ± SD from 3 independent experiments using Student’s t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Data in (D) are displayed as the mean ± SD from 3 independent experiments using one-way ANOVA tests (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

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As the activities of mitochondrial K⁺ channels or mPTP opening play key roles in regulating mitochondrial morphology [28], we employed their activators or inhibitors during serum starvation for 24 h. The mPTP facilitator mastoparan or K⁺ channel agonists, NS1619 (for K_Ca) and diazoxide (for K_ATP), all promoted donut formation. Conversely, the mPTP inhibitor cyclosporin A (CSA) or K⁺ channel inhibitors paxilline (for K_Ca) and 5-hydroxydecanoate (5HD, for K_ATP) prevented donut formation (Figure S2). These results indicate that the opening of mPTP or K⁺ channels are involved in accumulation of donut mitochondria under serum starvation, similar to what was observed with hypoxia-reoxygenation [28]. In addition, we observed that the K_Ca inhibitor prevented the formation of swollen mitochondria (Figure S2B), suggesting the dependence of K⁺ channel opening, which is consistent with the previous report of dose-dependent formation of donut and swollen mitochondria on K⁺ ionophores [28].

We employed transmission electron microscopy (TEM) to examine the mitochondrial ultrastructure. Using the ratio of the sum of mitochondrial cristae length to the mitochondrial area to represent the abundance of cristae, we found decreased cristae abundance in swollen mitochondria compared with donuts (Figure 1E,F). Following the fates of donut and swollen mitochondria under starvation, we observed that approximately 46% of donut mitochondria reverted to tubular mitochondria, whereas only a few swollen mitochondria exhibited the same fate (Figure 1G,H, Movie S1). These results suggest that the different changes in the topology of mitochondria under starvation result in different fates.

Mitophagy occurs in swollen but not donut mitochondria under starvation

As damaged mitochondria are selectively eliminated by autophagy under stress, we explored the relationship between mitochondrial topological transformations and mitophagy. We co-expressed mitochondrial matrix targeted DsRed (mtDsRed) and GFP-LC3B in U2OS cells and placed them under serum starvation for 24 h. We found that approximately 35% of swollen mitochondria colocalized with GFP-LC3B, indicating the presence of mitophagy, whereas none of donut mitochondria overlapped with GFP-LC3B (Figure 2A,B). Consistently, only swollen mitochondria stained with the HSPD1/HSP60 (heat shock protein family D member 1) antibody colocalized with autophagosomes stained with the LC3B antibody in the U2OS cells under 24-h serum starvation (Fig. S3A). As the fusion of autophagosome with lysosome is an important step of mitophagy, we analyzed the colocalization of lysosomes and mitochondria by co-expressing LAMP1 (lysosomal associated membrane protein 1)-GFP and mtDsRed in U2OS cells. Approximately 25% of swollen mitochondria were engulfed by lysosomes, but none of the donut mitochondria were sequestered by lysosomes after 24-h serum starvation (Figure 2C,D). Similar results were obtained by staining with HSPD1 and LAMP1 antibodies in U2OS cells under starvation (Fig. S3B). To further confirm the mitophagy process, we introduced mt-mKeima, a pH-sensitive, dual-excitation ratiometric fluorescent protein, to monitor the ongoing mitophagy [33]. Similarly, we also found that only swollen mitochondria were enveloped by lysosomes (Figure 2E,F and S4). In addition, TEM imaging confirmed the presence of mitophagy under serum starvation in U2OS cells (Figure 2G).

Topology-dependent, bifurcated mitochondrial quality control under starvation

All authors

Yanshuang Zhou, Qi Long, Hao Wu, Wei Li, Juntao Qi, Yi Wu, Ge Xiang, Haite Tang, Liang Yang, Keshi Chen, Linpeng Li, Feixiang Bao, Heying Li, Yaofeng Wang, Min Li & Xingguo Liu

https://doi.org/10.1080/15548627.2019.1634944

Published online:

04 July 2019

Figure 2. Mitophagy occurs in swollen but not donut mitochondria under serum starvation. (A) Representative images of U2OS cells expressing mtDsRed and GFP-LC3B after 24-h serum starvation. Scale bar: 5 μm. The boxed regions are magnified. Scale bar: 1 μm. (B) The percentage of swollen and donut mitochondria colocalized with GFP-LC3B in serum-starved U2OS cells. (C) Representative images of U2OS cells expressing mtDsRed and LAMP1-GFP after 24-h serum starvation. Scale bar: 5 μm. The boxed regions are magnified. Scale bar: 500 nm. (D) The percentage of swollen and donut mitochondria colocalized with LAMP1 in serum-starved U2OS cells. (E) Images of mt-mKeima at 458 (green) and 543 (red) nm excitations in U2OS cells after 24-h serum starvation. Scale bar: 5 μm. (F) Quantification of mt-mKeima signals in swollen and donut mitochondria in (E). (G) Representative TEM images of mitophagy after 24-h serum starvation in the presence of BAF in U2OS cells. The white arrowheads indicate that mitochondria were enveloped by autophagosome.Scale bar: 500 nm. Data in (B), (D) and (F) are shown as mean ± SD, and acquired from at least 150 cells from 3 independent experiments.

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It would be interesting to manipulate the levels of donut by morphology factors and detect their effects on mitophagy. As mitochondrial fission machinery is known to not be involved in donut formation [28,34], we determined the relationship between mitochondrial morphology and autophagy in mouse embryonic fibroblast (MEF) cells lacking the mitochondrial fusion factors MFN1 (mitofusin 1) and MFN2 (mitofusin 2). In mfn1 and mfn2 double-knockout (mfn1/2 KO) MEF cells, mitochondria were punctate with a large population of swollen mitochondria, but no donut mitochondria were observed (Figure S5A and S5B). Under serum starvation for 36 h, mfn1/2 KO MEF cells exhibited a higher level of mitophagy compared with wild-type (WT) MEF cells by FACS-based mt-mKeima assay (Figure S5C and S5D). These results are consistent with our hypothesis that donut mitochondria are protected from mitophagy.

To confirm that swollen mitochondria were selectively degraded by mitophagy, bafilomycin A₁ (BAF), an inhibitor of lysosome acidification and autophagosome-lysosome fusion, was used to prevent the digestion of mitochondria in autophagosomes [35]. Compared with the control, the number of swollen mitochondria significantly increased under BAF treatment. The percentage of swollen mitochondria colocalized with LC3B also increased obviously in the presence of BAF (Figure 3A,C,D). In parallel, the MTOR (mechanistic target of rapamycin kinase) inhibitor rapamycin was used to promote autophagy [35]. The number of swollen mitochondria showed no obvious change with rapamycin, whereas the percentage of swollen mitochondria colocalized with LC3B increased (Figure 3B–D). The number of donut mitochondria, however, showed no significant differences in the presence of rapamycin or BAF, and no colocalization of donuts with LC3B was observed under any of these conditions. To elucidate the role of canonical autophagy in this process, we performed serum starvation experiments in atg5 KO MEF cells (Figure 3E). No colocalization between mitochondria and LC3B was observed, but the number of swollen mitochondria increased in atg5 KO MEF cells compared with WT MEF cells (Figure 3F), indicating that that blocking autophagy induces the accumulation of swollen mitochondria. Taken together, these results imply that swollen but not donut mitochondria are targeted by ATG5-mediated autophagy for elimination.

Topology-dependent, bifurcated mitochondrial quality control under starvation

All authors

Yanshuang Zhou, Qi Long, Hao Wu, Wei Li, Juntao Qi, Yi Wu, Ge Xiang, Haite Tang, Liang Yang, Keshi Chen, Linpeng Li, Feixiang Bao, Heying Li, Yaofeng Wang, Min Li & Xingguo Liu

https://doi.org/10.1080/15548627.2019.1634944

Published online:

04 July 2019

Figure 3. Clearance of swollen mitochondria is dependent on ATG5-mediated autophagy flux under serum starvation. (A) Representative images of U2OS cells expressing mtDsRed and GFP-LC3B after 24-h serum starvation in the presence of BAF. Scale bar: 5 μm. The boxed regions are magnified. Scale bar: 500 nm. (B) Representative images of U2OS cells expressing mtDsRed and GFP-LC3B after 24-h serum starvation in the presence of rapamycin. Scale bar: 5 μm. The boxed regions are magnified. Scale bar: 1 μm. (C) The number of swollen and donut mitochondria per cell after 24-h serum starvation in the presence of BAF or rapamycin. (D) The percentage of swollen or donut mitochondria colocalized with GFP-LC3B after 24-h serum starvation in the presence of BAF or rapamycin. (E) Representative images of atg5 KO MEF cells expressing mtDsRed and GFP-LC3B after 24-h serum starvation. Scale bar: 5 μm. The boxed regions are magnified. Scale bar: 500 nm. (F) Quantification of swollen and donut mitochondria per cell under 24-h serum starvation in (E). Data in (C) and (D) are shown as mean ± SD, and acquired from 150–200 cells from 3 independent experiments using one-way ANOVA tests (**, p < 0.01; ***, p < 0.001). Quantification in (F) is shown as the mean ± SD from 3 independent experiments (>150 cells) using Student’s t-test (*, p < 0.05).

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δψ_m dissipation and PRKN recruitment occurs in swollen but not donut mitochondria under starvation

As mitochondria with low ΔΨ_m are selected for autophagy in a PINK1-PRKN-dependent manner [7–10], we asked whether the elimination of swollen mitochondria is also PINK1-PRKN dependent. First, we confirmed the presence of endogenous PRKN in U2OS cells by western blot and quantitative PCR (q-PCR) analysis (Figure S6). Human neuroblastoma SH-SY5Y cells were used as a positive control [10,36]. Then, we measured ΔΨ_m using tetramethylrhodamine methyl ester (TMRM). We found that, under starvation conditions, approximately 75% of swollen mitochondria had low ΔΨ_m, whereas most donut mitochondria maintained their ΔΨ_m (Figure 4A,B). To further study the relationship among mitochondrial morphology, ΔΨ_m and PRKN recruitment, we employed mtDsRed, YFP-PRKN and 1, 1ʹ, 3, 3, 3ʹ, 3ʹ-hexamethylindodicarbo-cyanine iodide (DiIC1(5)) (an indicator of ΔΨ_m) [37]. As expected, swollen mitochondria lost their ΔΨ_m and recruited PRKN, whereas donut mitochondria maintained their ΔΨ_m and did not recruit PRKN (Figure 4C,D). Next, we labeled U2OS cells with mtDsRed, GFP-LC3B and DiIC1(5), and similar results were observed, wherein LC3B was recruited to the swollen mitochondria with ΔΨ_m dissipation (Figure 4E). Taken together, these results demonstrate that ΔΨ_m dissipation and subsequent PRKN recruitment occurs in swollen mitochondria but not in donuts under serum starvation.

Topology-dependent, bifurcated mitochondrial quality control under starvation

All authors

Yanshuang Zhou, Qi Long, Hao Wu, Wei Li, Juntao Qi, Yi Wu, Ge Xiang, Haite Tang, Liang Yang, Keshi Chen, Linpeng Li, Feixiang Bao, Heying Li, Yaofeng Wang, Min Li & Xingguo Liu

https://doi.org/10.1080/15548627.2019.1634944

Published online:

04 July 2019

Figure 4. ΔΨ_m dissipation and PRKN recruitment occur in swollen but not donut mitochondria under serum starvation. (A) Representative images of U2OS cells expressing mtGFP with TMRM staining under serum starvation. Line scans on the right indicate colocalization between mtGFP and TMRM and correspond to the lines drawn in the images. Scale bar: 5 μm. The boxed regions are magnified. Scale bar: 1 μm. (B) Quantification of ΔΨ_m of swollen and donut mitochondria in U2OS cells under 24-h serum starvation. (C) Representative images of U2OS cells labeled with mtDsRed, YFP-PRKN and DiIC1(5) after 24-h serum starvation. Line scans on the right indicate the colocalization among mtDsRed, YFP-PRKN and DiIC1(5) and correspond to the lines drawn in the images. Scale bar: 5 μm. The boxed regions are magnified. Scale bar: 1 μm. (D) The percentage of swollen or donut mitochondria with PRKN recruitment in (C). (E) Representative images of U2OS cells labeled with mtDsRed, GFP-LC3B and DiIC1(5) after 24-h serum starvation. Line scans on the right indicate the colocalization among mtDsRed, GFP-LC3B and DiIC1(5) and correspond to the lines drawn in the images. Scale bar: 5 μm. The boxed regions are magnified. Scale bar: 1 μm. (F) Relative expression level of PINK1, PRKN, CALCOCO2, OPTN and ULK1 from U2OS cells under serum starvation for the indicated time. The samples at 0 h were used as a control to which gene expression levels were normalized (n = 3). (G) Protein levels of LC3B-I, LC3B-II, TOMM20, PINK1, PRKN, CALCOCO2, OPTN and ULK1 from U2OS cells under serum starvation for the indicated time (n = 3). Data in (B), (D) and (F) are shown as mean ± SD, and in (B) and (D) are at least 100 cells from 3 independent experiments.

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To further determine the dependence of mitophagy on the PINK1-PRKN pathway under serum starvation, we knocked down PINK1 in U2OS cells. We detected a decrease in mitophagy in PINK1 knockdown (KD) cells under serum starvation for 36 h using the mt-mKeima FACS assay (Figure S7A–S7C). Next, we examined autophagy receptors, downstream of the PINK1-PRKN pathway, CALCOCO2, OPTN and ULK1, which would be recruited to mitochondria to promote mitophagy [13]. The q-PCR and western blot analysis showed the upregulation of PINK1, PRKN, CALCOCO2, OPTN and ULK1 under serum starvation in U2OS cells (Figure 4F,G). Meanwhile, a decline of TOMM20 (translocase of outer mitochondrial membrane 20) and accumulation of LC3B-II were also observed under serum starvation (Figure 4G). These results indicate that mitophagy under serum starvation is mediated by the PINK1-PRKN pathway.

Depolarized donut mitochondria resist autophagy even after PRKN recruitment and ubiquitination

To determine whether donut mitochondria would be eliminated upon loss of ΔΨ_m. U2OS cells labeled with mtDsRed and YFP-PRKN were exposed to 10 μM FCCP for 0, 0.5, 1 and 5 h. (Figure 5A). Mitochondria rapidly responded to the treatment with approximately 30% becoming donuts at 0.5 h. However, the proportion of donut mitochondria declined again to 0.3% at 5 h treatment, whereas the presence of swollen mitochondria increased continuously over that time (Figure 5B). Both depolarized donuts and swollen mitochondria recruited PRKN after 1-h FCCP treatment (Figure 5C,D). To verify whether depolarized donut mitochondria would be eliminated after PRKN recruitment, the colocalization of mitochondria and autophagosomes (marked with GFP-LC3) was analyzed. Swollen mitochondria overlapped with GFP-LC3B dots whereas donut mitochondria that recruited PRKN never colocalized with GFP-LC3B (Figure 5E,F). As for the fate of donuts after FCCP treatment, we observed that a donut mitochondrion separated into 2 mitochondria of different shapes: one swollen and one fragmented (Figure 5G, Movie S2). These results suggest that donut mitochondria could still resist autophagy even after the recruitment of PRKN.

Topology-dependent, bifurcated mitochondrial quality control under starvation

All authors

Yanshuang Zhou, Qi Long, Hao Wu, Wei Li, Juntao Qi, Yi Wu, Ge Xiang, Haite Tang, Liang Yang, Keshi Chen, Linpeng Li, Feixiang Bao, Heying Li, Yaofeng Wang, Min Li & Xingguo Liu

https://doi.org/10.1080/15548627.2019.1634944

Published online:

04 July 2019

Figure 5. Depolarized donut mitochondria resist autophagy even after PRKN recruitment. (A) Representative images of U2OS cells expressing mtDsRed and YFP-PRKN treated with FCCP for the indicated time. Scale bar: 5 μm. (B) The percentages of swollen and donut mitochondria per cell in FCCP treated U2OS cells for the indicated time. (C) Representative images of U2OS cells expressing mtDsRed and YFP-PRKN after FCCP treatment for 1 h. Scale bar: 5 μm. The boxed regions are magnified. Scale bar: 1 μm. (D) The percentages of swollen and donut mitochondria with PRKN recruitment in FCCP treated U2OS cells for the indicated time. (E) Representative images of U2OS cells expressing mtDsRed and GFP-LC3B treated with FCCP for 1 and 5 h, respectively. Scale bar: 5 μm. The boxed regions are magnified. Scale bar: 1 μm. (F) Quantification of swollen or donut mitochondria colocalized with LC3B in FCCP treated U2OS cells. (G) Time series of a cell expressing mtDsRed and YFP-PRKN in U2OS cells after 1-h FCCP treatment. The white arrowheads indicate a donut mitochondrion splitting into mitochondria of different shapes after PRKN recruitment. Scale bar: 2 μm. Data in (B), (D) and (F) are shown as mean ± SD, and acquired from100–200 cells from 3 independent experiments.

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To further determine the mechanism by which donuts resist autophagy under depolarization, we detected the mitochondrial ubiquitination and the recruitment of the autophagy receptors CALCOCO2 and OPTN. Both swollen and donut mitochondria showed the presence of poly-ubiquitination after 2-h FCCP treatment in U2OS cells (Figure 6A,B). However, CALCOCO2 recruitment occurred mainly on swollen mitochondria but rarely on donuts (Figure 6C,D). We observed similar results for OPTN recruitment (Figure 6E,F). Taken together, these results demonstrate that donuts’ resistance to autophagy under depolarization is due to the prevention of mitochondria from recruiting the autophagosome receptors CALCOCO2 and OPTN even after PRKN recruitment and ubiquitination.

Topology-dependent, bifurcated mitochondrial quality control under starvation

All authors

Yanshuang Zhou, Qi Long, Hao Wu, Wei Li, Juntao Qi, Yi Wu, Ge Xiang, Haite Tang, Liang Yang, Keshi Chen, Linpeng Li, Feixiang Bao, Heying Li, Yaofeng Wang, Min Li & Xingguo Liu

https://doi.org/10.1080/15548627.2019.1634944

Published online:

04 July 2019

Figure 6. CALCOCO2 and OPTN recruitment occur in swollen but few in donut mitochondria under FCCP treatment. (A) Immunostaining for polyubiquitinated protein of U2OS cells expressing mtDsRed and YFP-PRKN treated with FCCP for the indicated time. Scale bar: 5 μm. (B) The percentage of swollen or donut mitochondria with ubiquitin recruitment in (A). (C) Representative images of U2OS cells expressing mtTFP, mCherry-CALCOCO2 and YFP-PRKN treated with FCCP for the indicated time. Scale bar: 5 μm. (D) The percentage of swollen or donut mitochondria with CALCOCO2 recruitment in (C). (E) Representative images of U2OS cells marked with mtDsRed and GFP-OPTN were treated with FCCP for the indicated time. Scale bar: 5 μm. (F) The percentage of swollen or donut mitochondria with OPTN recruitment in (E). (G) Model of topological control of mitophagy. Data in (B), (D) and (F) are shown as the mean ± SD, and acquired from at least 1000 mitochondria in 3 independent experiments. Data in (D) are analyzed using Student’s t-test (***, p < 0.001).

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Cell culture

U2OS (ATCC, HTB-96), HEK 293T (ATCC, CRL-3216), atg5^−/− MEF (a gift from Prof. Min Li, Sun Yat-Sen University), mfn1/2^−/− MEF (ATCC, CRL-2994), WT MEF (ATCC, CRL-2991), SH-SY5Y (ATCC, CRL-2266) and SHG44 (RRID:CVCL_6728) cells were cultured in Dulbecco modified Eagle medium (DMEM; Hyclone, SH30022.01) supplemented with 10% (v/v) fetal bovine serum (Industria Argentina, NTC-HK008), GlutMAX (Gibco, 35,050–061) and NEAA (Gibco, 11,140–050). All cells were cultured in cell incubator (Thermo Scientific, 3111) containing 5% CO₂ at 37°C.

Plasmids

The plasmid pDsRed2-mito (Clontech, 632,421) was used to mark mitochondria with a mitochondrial targeting sequence from COX8A (cytochrome c oxidase subunit 8A) fused to the fluorescence protein DsRed2. pMXs-mtDsRed was constructed by inserting pDsRed2-mito into the pMXs-flag vector. By replacing DsRed2 with GFP or TFP, we produced pMXs-mtGFP and pMXs-mtTFP. LAMP1 fused with GFP was also cloned into pMXs-Flag as a lysosome tracker. mt-mKeima and YFP-PRKN were described in our previous report [54]. CALCOCO2 and OPTN fused with GFP or mCherry were cloned into pMXs-Flag. pCL plasmid (Imgenex, 10045P) was used to code the virus package elements. shRNA for PINK1 was constructed into the pLKO.1-puro empty vector. The target sequence for PINK1 silencing is CGGCTGGAGGAGTATCTGATA. All plasmids used in this study are listed in the Table S1.

Generation of stable cell lines

Stable cell lines were generated by the infection of retroviruses containing mtDsRed, mtGFP, mtTFP, LAMP1-GFP, GFP-LC3B, mt-mKeima, YFP-PRKN, mCherry-CALCOCO2, or GFP-OPTN. Briefly, retroviruses were produced by transfection of HEK 293T cells with the expression plasmids by polyethyleneimine (Polysciences, 23,966) method. Retroviruses were collected and filtered with 0.45-μm filters (Millipore, SLHV033RB) 48 h after transfection and then used to infect U2OS cells or MEFs with polybrene (Sigma, H9268) to generate stable cell lines. The KD pLKO.1-puro vectors (Sigma, SHC016-1EA) were co-transfected with helper plasmids psPAX2 and pMD2.g into HEK293T cells. Lentiviruses collection and transfection was carried out the same as retroviruses. Cells infected with pLKO.1-puro were screened with puromycin (Genomeditech, GM-040401–2; 2 μg/mL for 48 h).

Serum starvation and drug treatments

Cells were plated at 70% confluence on a 35-mm dish (WPI, FD35-100) coated with 0.1% gelatin (Millipore, ES-006-B). For serum starvation, the culture medium was replaced with serum-free medium for different periods as indicated. FCCP (10 μM; Sigma, C2920) or rapamycin (100 nM; MedChemExpress, HY-10,219) was added to the medium during serum starvation. These treatments were also performed in the presence of CSA (1 μM; Santa Cruz Biotechnology, sc-3503), mastoparan (0.5 μM; Sigma, C3662-5MG), diazoxide (10 μM; Sigma, D9035), NS1619 (3 μM; Abcam, ab141824), 5HD (500 μM; Sigma, H135) or paxilline (1 μM; Alomone Labs, P-450). Cells were treated with BAF (100 nM; Gene Operation, C2501) for 3 h prior to imaging to prevent autophagosome-lysosome fusion after serum starvation.

TEM

As in our previous report [54], cultured cells were fixed with 3.0% glutaraldehyde (Sigma, G5882) for more than 12 h at 4°C after washing with PBS (Genom, GNM20012). The samples were fixed with 1% osmium acid (SPI-Chem, 02601-AB) for another 1 h before dehydration by gradient ethanol (Sigma, E7023). Following the typical TEM sample preparation procedure, the samples were stained with lead (SPI-Chem, 02532-AB) and uranium (SPI-Chem, 02624-AB) after ultra-sectioning with Leica EM UC7 (Germany). The ultrastructure of mitochondria and autophagosomes were visualized by TEM (FEI Tecnai G2 Spirit, USA) in 120 KV.

Immunofluorescence

U2OS cells, seeded in coverslips (Thermo, CB00130RAC20MNT0), were fixed for 10 min at room temperature with fixative containing 3% paraformaldehyde (Genesion, JX0100) and 0.1% glutaraldehyde. Then, cells were permeabilized with 0.5% Triton X-100 (Sigma, T8787) for 5 min and blocked with 10% goat serum (Jackson, 005–000-121) in PBS for 1 h at room temperature. For immunostaining, cells were incubated with antibodies (as indicated in the figure legends) diluted in 5% goat serum buffer overnight at 4°C and rinsed with PBS. Alexa Fluor 488 or 568 conjugated secondary antibodies (Life Technologies, A-11,008, A-11,004) were used for confocal imaging. Cells were washed 5 times with PBS. During the final washing step, cells were incubated with DAPI (10 μg/mL; Sigma, D9542) in PBS for 5 min. The detailed information of primary antibodies used in this study is shown in Table 1.

δψ_m measurement

For TMRM staining, cells were stained with 25 nM TMRM (Invitrogen, T668) for 30 min and imaged in the presence of 5 nM TMRM. For DiIC1(5) (Molecular Probes, M34151) imaging, cells were stained with 50 nM DiIC1(5) for 25 min, and then washed twice before imaging.

Western blotting

Cells were lysed in radioimmunoprecipitation (Beyotime, P0013B) buffer, containing protease inhibitor cocktail (Roche, 04693116001) and phenylmethanesulfonyl fluoride (Beyotime, ST506). The detailed information of primary antibodies used in this study is shown in Table 1. HRP-conjugated goat anti-rabbit antibody (Kangchen, KC-RB-035) and goat anti-mouse antibody (Kangchen, KC-MM-035) were used in this assay. We used ECL (Merck Millipore, WBKLS0500) to detect the target proteins.

Reverse transcription-PCR (RT-PCR) and q-PCR

Cells were lysed with TRIzol reagent (Molecular Research Center, TR118) after washing twice with PBS. Total RNA was extracted following the manufacturer’s protocol. The cDNA synthesis was performed with RT-PCR Kit (Takara, 639,522). The q-PCR was performed with a real-time PCR-SYBR Green kit (Takara, RR430S), and run on CFX Connect (Bio-Rad, USA). The human ACTB was used as a control. The q-PCR primers we used in this study are shown in Table S2.

Mt-mKeima mitophagy assay

WT and PINK1 KD U2OS cells stably expressing mt-mKeima were generated using lentiviral transduction as described above. WT MEF and mfn1/2 KO MEF cells stably expressed mt-mKeima by retroviral transduction, as described above. The cells were then subjected to serum starvation for 36 h. Subsequently, they were resuspended in sorting buffer (145 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 10 mM HEPES, pH 7.4, 10 mM glucose, 0.1% BSA [MultiSciences, A3828-100]) containing 10 μg/mL DAPI. Analysis was performed using Summit software on a Beckman Coulter MoFlo Astrios cell sorter (USA). Measurements of lysosomal mt-mKeima were made using dual-excitation ratiometric pH measurements at 488 (pH 7) and 561 (pH 4) nm lasers with 620/29 nm and 614/20 nm emission filters, respectively. For each sample, 50,000 events were collected and subsequently gated for mt-mKeima-positive cells that were DAPI-negative. Data were analyzed using FlowJo (v10, Tree Star).

Live cell imaging

Cells were imaged using a Zeiss LSM 710 inverted confocal laser scanning microscope (Germany) in 37°C, 5% CO₂ and humidity control system. GFP and YFP were excited with a 488-nm laser, whereas DsRed2 and TMRM were exited with a 543-nm laser. DiIC1(5) was excited with a 633-nm laser. The mKeima was excited with a 458-nm and a 543-nm laser. Images were taken at 1024 × 1024 pixel with a Plan Apo 100× oil lens (Zeiss, N.A. = 1.40). The images were analyzed with Zen2012 (Zeiss), ImageJ [55], AutoQuant X3 (Bitplane) and Imaris v7.6 (Bitplane) software.

Quantification of donut and swollen mitochondria

Numbers of donut and swollen mitochondria were manually counted in each cell. The standard of donut mitochondria was described in our previous report [28]. Only mitochondrial structures containing a central hole were classified as donut mitochondria, whereas swollen mitochondria were simply round shaped. Acquisition of z-series and 3D reconstruction confirmed that swollen mitochondria were spheroid (Figure S8). To distinguish swollen mitochondria from fragmented mitochondria, we defined swollen mitochondria as those with a diameter of at least twice the diameter of tubular mitochondria. Mitochondrial diameter is ∼200–300 nm in MEF cells as our previous report [47].

Cell viability analysis

Trypan blue staining was used to assess cell viability. U2OS cells were subjected to serum starvation for the indicated time. Then, cells were suspended and stained using the Trypan Blue Staining Kit (Beyotime, C0011) according to the manufacturer’s instructions to measure cell viability.

Statistical analysis

Statistical analysis was conducted using GraphPad PRISM. Data are shown as mean ± SD. To assess statistical significance, data from 3 independent experiments were analyzed using one-way ANOVA and Tukey’s post-test or two-tailed unpaired Student’s t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).