Parkin-dependent mitophagy by preventing mitochondrial Parkin accumulation

Journal Pre-proof Bcl-xL inhibits PINK1/Parkin-dependent mitophagy by preventing mitochondrial Parkin accumulation Si Yu, Mengyan Du, Ao Yin, Zihao Mai, Yong Wang, Mengxin Zhao, Xiaoping Wang, Tongsheng Chen

PII:

S1357-2725(20)30037-6

DOI:

https://doi.org/10.1016/j.biocel.2020.105720

Reference:

BC 105720

To appear in:

International Journal of Biochemistry and Cell Biology

Received Date:

26 November 2019

Revised Date:

18 February 2020

Accepted Date:

19 February 2020

Please cite this article as: Yu S, Du M, Yin A, Mai Z, Wang Y, Zhao M, Wang X, Chen T, Bcl-xL inhibits PINK1/Parkin-dependent mitophagy by preventing mitochondrial Parkin accumulation, International Journal of Biochemistry and Cell Biology (2020), doi: https://doi.org/10.1016/j.biocel.2020.105720

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Bcl-xL inhibits PINK1/Parkin-dependent mitophagy by preventing mitochondrial Parkin accumulation Si Yua, Mengyan Dua, Ao Yina, Zihao Maia, Yong Wanga, Mengxin Zhaob, Xiaoping Wangb,*, Tongsheng Chena,** a

MOE Key Laboratory of Laser Life Science & Guangdong Provincial Key

Laboratory of Laser Life Science, College of Biophotonics, South China Normal

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University, Guangzhou 510631, China Department of Pain Management, the First Affiliated Hospital, Jinan University,

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Guangzhou 510632, China

* Correspondence to: X. Wang, Department of Pain Management, the First

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Affiliated Hospital, Jinan University, Guangzhou 510632, China.

** Correspondence to: MOE Key Laboratory of Laser Life Science &

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Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.

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E-mail addresses: [email protected] (X. Wang); [email protected] or

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[email protected] (T. Chen).

Highlights

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Bcl-xL inhibits PINK1/Parkin-dependent mitophagy.

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Bcl-xL prevents mitochondrial Parkin accumulation.

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Bcl-xL physically interacts with Parkin in cytoplasm.

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Bcl-xL physically interacts with PINK1 on mitochondria.

Abstract

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This report aims to explore how Bcl-xL, a Bcl-2 family protein, regulates PINK1/Parkin-dependent mitophagy. Compared with the Hela cells expressing Parkin alone, co-expression of Bcl-xL significantly inhibited CCCP (Carbonyl cyanide 3chlorophenylhydrazone)-induced mitochondrial Parkin accumulation and mitophagy. Western blotting analysis illustrated that over-expressed Bcl-xL inhibited CCCP-induced decrease of mitochondrial proteins in Parkin over-expressed cells. Fluorescence loss in photobleaching (FLIP) analyses demonstrated that Bcl-xL inhibited the CCCP-induced translocation of Parkin into mitochondria not by retrotranslocating Parkin from mitochondria to cytoplasm. Fluorescence resonance energy transfer (FRET) imaging revealed in Hela cells that Bcl-xL physically bound with Parkin to form oligomer in cytoplasm, and that Bcl-xL also directly interacted with PINK1 on mitochondria. Co-immunoprecipitation analysis for HEK293T cells verified that endogenous Bcl-xL interacted with both endogenous Parkin and PINK1. Collectively, Bcl-xL inhibits PINK1/Parkin- dependent mitophagy by preventing the accumulation of Parkin on mitochondria via two regulation ways: directly binds to Parkin in cytoplasm to prevent the translocation of Parkin from cytoplasm to mitochondria and directly binds to PINK1 on mitochondria to inhibit the recruitment of Parkin from cytoplasm to mitochondria by PINK1.

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Keywords: Bcl-xL; Parkin; PINK1; mitophagy; FRET; living cells.

1. Introduction

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Autophagy can degrade various cytoplasmic components (Chen and Klionsky, 2010; Mizushima, 2007) and can also eliminate damaged organelles (Narendra et al., 2010; Okatsu et al., 2010) by autolysosomes. Mitophagy is the main mechanism of eliminating damaged mitochondria in both yeast and mammals (Kanki and Klionsky, 2010; Kim et al., 2007). Mitophagy, a organelle-specific autophagy, was first observed in mammalian cells by early electron microscopy studies (De Duve and Wattiaux, 1966), and refers to the selective elimination of mitochondria by autophagy (Lemasters, 2005). Different kinds of stress, such as oxidative damage, mitochondrial depolarization and mitochondrial DNA (mtDNA) damage, can activate mitophagy (Eid et al., 2016; Gorgoulis et al., 2018). More and more proteins have been identified to participate in mitophagy (Dagda et al., 2008; Melser et al., 2013; Shiba-Fukushima et al., 2012; Wu et al., 2014). PINK1 and Parkin have been demonstrated to regulate mitophagy (Pickrell and Youle, 2015). PINK1, a mitochondrial Ser/Thr kinase, was identified as relatively gene for primary Parkinsonism (Valente et al., 2004). In healthy mitochondria, PINK1 is anchored on the mitochondrial outer membrane and quickly cleaved by mitochondrial proteases (such as MPP, PARL, ClpXP and AFG3L2) to form two smaller isoforms, resulting in the retranslocation of N-PINK1 isoform to cytoplasm and subseqent degradation by ubiquitin proteasome (Greene et al., 2012; Jin et al., 2010; Muqit et al., 2006). However, dissipated mitochondrial membrane potential (ΔΨm) result in the localization of the full-length PINK1 on the damaged mitochondrial membrane where full-length PINK1 accumulates and autophosphorylates to recruit Parkin from cytoplasm to mitochondria (Okatsu et al., 2013). Parkin, an E3 ubiquitin ligase protein, is repressed under normal conditions (Kitada, 1998). Activited PINK1 triggers phosphorylation of Parkin and increases mitochondrial accumulation of Parkin (Kane et al., 2014; Koyano et al., 2014). Mitochondrial Parkin triggers mitophagy by polyubiquitinating mitochondrial outer membrane proteins (de Vries and Przedborski, 2013; Kondapalli et al., 2012; Okatsu et al., 2012; Pickrell and Youle, 2015). PINK1 functions as a sensor to accept collapsing ΔΨm, while Parkin acts as an effector to polyubiquitinate various proteins, then activate mitophagy. Bcl-2 family members participate in the regulation of mitophagy (Gomes et al., 2011; Hollville et al., 2014; Rambold et al., 2011; Twig et al., 2008). The interaction between anti-apoptotic Bcl-2 family proteins and autophagic protein Beclin1 can regulate macroautophagy (Erlich et al., 2007; Pattingre et al., 2005). It has been reported that and BNIP3 proteins were required for mitophagy when cells are in hypoxia (Novak, 2012; Thomas et al., 2011; Zhang and Ney, 2009). Hollville and colleagues (Hollville et al., 2014) reported that Bcl-2 family proteins (including anti-apoptosis proteins, pro-apoptosis proteins and BH3-only proteins) regulated PINK1/Parkin-dependent mitophagy. Although this research elegantly demonstrated that Bcl-xL inhibited PINK1/Parkin-dependent mitophagy, the molecular mechanisms underlying remain poorly understood.

2. Material and methods

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2.1 Eukaryotic expression vectors, siRNA and antibodies

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This report aims to investigate how Bcl-xL regulates the PINK1/Parkin-dependent mitophagy by using fluorescence imaging technology including fluorescence resonance energy transfer (FRET) and fluorescence loss in photobleaching (FLIP). Fluorescence imaging exhibited that Parkin was even distribution in the cells co-expressing Bcl-xL and Parkin, and FLIP analyses demonstrated that Parkin did not undergo retrotranslocation from mitochondria to cytoplasm by Bcl-xL. FRET analyses discovered that Bcl-xL interacted with both Parkin and PINK1: Bcl-xL physically bound to Parkin in cytoplasm to prevent the mitochondrial translocation of Parkin; Bcl-xL interacted with PINK1 on mitochondria to inhibit the recruitment of Parkin by PINK1, which was also confirmed by co-immunoprecipitation analysis on endogenous Bcl-xL, Parkin and PINK1 in HEK293T cells. Our findings provide insight into regulation mechanism between Bcl-xL and PINK1/Parkin during mitophagy.

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CFP-Bcl-xL plasmid (Valentijn and Gilmore, 2004) and four standard plasmids (C4Y, C10Y, C40Y and C80Y) (Butz et al., 2016) were shared by Prof. Gilmore and Prof. Wahl-Schott. CFP (#13030), YFP (#13033), CFP-Parkin (#47560) and PINK1 C-GFP (#13316) plasmids were purchased from Addgene company (Cambridge, Massachusetts). Full-length PINK1 was amplified from PINK1-GFP using PCR and was subcloned into the pEYFP-C1-Drp1 (Addgene, #45160) plasmid by double enzyme digestion in the XhoI and BamHI sites. A pool of three siRNA against PINK1 1: (5'- CGA AGC CAU CUU GAA CAC AAU-3'), 2: (5'-GCC GCA AAU GUG CUU CAU CUA-3'), and 3: (5'-GUU CCU CGU UAU GAA GAA CUA-3') was purchased from igeBio (Guangzhou, China). The primary antibodies of anti-Parkin (#4211, CST), anti-PINK1 (#6946, CST), anti-Bcl-xL (#2764, CST) and anti-Tom20 (#42406, CST were used at 1:1000 and anti-GAPDH (K200057M, Solarbio) was used at 1:3000 for immunoblotting. Anti-Tom20 (#42406, CST) was used at 1:500 for immunofluorescence. Secondary antibodies connected with Alexa Fluor 680 and Alexa Fluor 780 for Western blotting experiments were respectively purchased from Invitrogen and Jackson (Massachusetts, USA; Pennsylvania, USA). 2.2 Cell culture，staining and DNA or siRNA transfection The source and culture method of Hela cells were just described previously (Zhang et al., 2016). HEK293T cells were a generous gift from Sino-French Hoffmann Institute, Guangzhou Medical University (Guangzhou, China) and cultured in the same method as Hela cells. For staining experiments, the dye of Mitotracker Deep Red 633 (Thermo Scientific) or DiIC1(5) (Invitrogen, Carlsbad, USA) was mixed with culture

medium and incubated with cells according to the protocol provided by manufacture to label mitochondria and mitochondrial membrane potential. Cells were sowed in 6-well plates or sowed in confocal dish and cultured in DMEM containing 10% FBS overnight. When the density reached to 70-80%, the cells were transfected with corresponding plasmid using Turbofect Transfection Reagent (Thermo Scientific) according to the instructions provided by manufacturer. The siRNA (100 nM) transfection was performed using Lipofectamine 2000 (Invitrogen, CA) following the guidelines of manufacture. A negative scrambled siRNA (igeBio, Guangzhou, China) was used in parallel. 2.3 Immunofluorescence and Western blotting analysis

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2.4 Quantitative FRET measurement methods

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For immunofluorescence experiments, cells were cultured on confocal dish for overnight and transfected with plasmid over 24 hr, then treated with Carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma-Aldrich, USA) for indicated time, and the following process of immunofluorescence experiments was performed just as described previously (Wang, B. et al., 2019). Western blotting analysis was performed as described previously (Wang, L. et al., 2019).

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Donor-centric FRET efficiency (ED) and acceptor/donor concentration ratio (RC) were obtained by E-FRET method as described previously (Du et al., 2018; Erickson et al., 2001). FRET efficiency and acceptor/donor concentration ratio were measured by acquiring the DD, DA and AA raw images by using different excitation/emission filters: DD: Fluorescence in Donor detection channel (BP480/40 nm) during Donor excitation (BP436/20 nm); DA: Fluorescence in Acceptor detection channel (BP535/30 nm) during Donor excitation (BP436/20 nm); AA: Fluorescence in Acceptor detection channel (BP535/30 nm) during Acceptor excitation (BP500/20 nm).

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2.5 Living-cell fluorescence imaging Quantitative FRET measurements were executed on an inverted ApoTome fluorescence microscope (Carl Zeiss, Oberkochen, Germany) as described previously (Wang, B. et al., 2019). Living-cell fluorescence imaging was also done on the ApoTome fluorescence microscope or a wide-field fluorescence microscope (Olympus IX73 equipped with a CCD camera, Japan). The excitation and emission filter used for imaging of CFP and YFP were described previously (Zhang et al., 2019). 550 nm excitation and 585/20 nm emission filter (Chroma) were used for RFP

imaging. 630 nm excitation and 660/20 nm emission filter (Chroma) were used for Mitotracker Deep Red or DiIC1(5) imaging. 2.6 FLIP assays FLIP experiments were performed on a confocal microscope (Zeiss LSM 880, Germany) as described previously (Edlich et al., 2011; Wang, B. et al., 2019). In brief, a single YFP region (diameter of 8 µm) was repeatedly bleached with twenty iterations by the maximal 514 nm laser. Two images were taken after each bleach pulse of 30 s apart. Fluorescence on mitochondria was measured separately after each cycle of bleaching for YFP-FLIP. The neighboring cells unbleached acted controls to monitor photobleaching during image acquisition.

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2.7 Immunoprecipitation assay

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HEK293T cells were lysed in RIPA lysis buffer (50mM Tris(pH 7.4)，150mM NaCl， 1% NP-40，0.5% sodium deoxycholate，0.1% SDS) (Beyotime, Shanghai, China) containing protease inhibitors (EpiZyme, Shanghai, China). The cells were lysed in the ice for 20 min, centrifugated at 13000 × g for 10 min at 4 ℃ and taken the supernatant. The co-immunoprecipitation was performed by the protocol of protein A/G magnetic beads (MCE, New Jersey, USA). 2.8 Statistics

3. Results

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Statistical analyses and histograms graphed were carried out by using the software of SPSS17.0 (SPSS, Chicago) and Origin 8.0 (Origin Lab Corporation), respectively.

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3.1 CCCP induces PINK1/Parkin-dependent mitophagy

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Microscopic imaging was used to inspect the location of Parkin and morphology of mitochondria after CCCP treatment. Cells were transfected with CFP-Parkin for 24 hr before being treated with CCCP and labeled mitochondria by immunostaining of TOM20. Parkin was even distribution and mitochondria were tubular in the cells without CCCP treatment (Fig. 1A; first row). CCCP treatment for 6 hr significantly induced mitochondrial localization of Parkin and mitochondrial networks collapsed around the perinuclear region (Fig. 1A；second row). In contrast to the even distribution of Parkin in the cells expressing CFP-Parkin without CCCP treatment, CFP-Parkin translocated from cytoplasm to mitochondria in about 90% of the Parkin-expressed cells after 6 hr CCCP treatment. After 24 hr CCCP treatment, about 81% of cells expressing CFP-Parkin exhibited even distribution of Parkin again and

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loss of TOM20 (Fig. 1A；third row), indicating that CCCP induces mitochondrial translocation of Parkin and subsequent mitophagy. Statistical results on the percentages of cells with cytosolic and mitochondrial Parkin distribution from at least 100 cells were shown in Figure 1B, and the corresponding results on CCCP-induced loss of TOM20 were shown in Figure 1C. In order to prove whether Parkin is necessary for mitochondrial clearance in response to CCCP treatment, cells were transfected with empty vector as control. Unlike to the Parkin-expressed cells, the cells expressed empty vector failed to eliminate mitochondria after 6 hr CCCP treatment (Fig. 1A; last line). Figure 1C showed the statistical result of cells with TOM20 (+) or TOM20 (-). After 6 hr CCCP treatment, about 95% of cells expressing CFP-vector exhibited TOM20 (+) but 18% of cells expressing CFP-Parkin exhibited TOM20 (+), indicating that Parkin is essential for mitophagy. To further verify the pattern of loss in mitochondrial protein, cells were co-transfected with CFP-Parkin and RFP-LC3 for 24 hr. In the cells co-expressing Parkin and LC3, contrast to the even distribution of both Parkin and LC3 in control cells, CCCP treatment for 6 hr induced co-localization of the two proteins with mitochondria (Fig. 1D). Figure 1E shows the statistical results about the punctate LC3 distribution in Parkin- and LC3-expressed cells at the indicated CCCP treatment times. CCCP treatment for 6 hr increased the proportions of cells with punctate LC3 from 6% (control) to 77% (cells co-expressing Parkin and LC3), further demonstrating that CCCP treatment induces autophagy in Parkin-transfected cells. We also inspected the role of PINK1 in the CCCP-induced mitochondrial recruitment of Parkin. The level of PINK1 in the cells knockdown by three independent small interfering RNAs (siRNAs) was detected by Western blotting analysis (Fig. S1). The siPINK1#2 was used for knockdown of PINK1 in this report. The cells co-transfected with scrambled (Scr) siRNA (control) or PINK1 siRNA and CFP-Parkin for 48 hr were treated with CCCP for 6 hr before microscopic imaging. As expected, PINK1 knockdown blocks the CCCP-induced accumulation of CFP-Parkin on to mitochondria (Fig. 1F). Figure 1G showed the statistical proportion of cells with mitochondrial Parkin. After 6 hr CCCP treatment, about 80% of cells expressing CFP-Parkin and co-transfected with Scr siRNA exhibited mitochondrial Parkin, but only 35% of cells expressing CFP-Parkin and co-transfected with siPINK1 exhibited mitochondrial Parkin, proving that PINK1 is essential for the mitochondrial recruitment of Parkin. 3.2 Bcl-xL inhibits PINK1/Parkin-dependent mitophagy To assess the effect of Bcl-xL in CCCP-induced collapse of ΔΨm, CFP-Bcl-xL was transfected into cells for 24 hr before CCCP treatment for 0 hr or 20 hr. Before fluorescence imaging, cells were stained with Mitochondrial membrane potential dye DiIC1(5). CCCP treatment for 20 hr induced loss of DiIC1(5) fluorescence in cells expressing CFP and CFP-Bcl-xL (Fig. 2A). Figure 2B showed the statistical results of cells with negative DiIC1(5). In control cells, about 3% of cells exhibited negative

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DiIC1(5), but in CCCP-treated cells, about 75% of the cells exhibited negative DiIC1(5), indicating that CCCP induced loss of mitochondrial membrane potential independent of Bcl-xL. In order to survey whether Bcl-xL was involved in PINK1/Parkin-dependent mitophagy, cells over-expressing CFP-Parkin and YFP or YFP-Bcl-xL were treated with CCCP for 24 hr. Mitochondria was labeled by immunostaining for Tom20. Fluorescence images showed that TOM20 almost disappeared in the cells co-expressing CFP-Parkin and YFP, but TOM20 still existed in the cells co-expressing CFP-Parkin and YFP-Bcl-xL (Fig. 2C). Statistical results from at least 100 cells showed that CCCP treatment for 24 hr induced loss of TOM20 in about 80% of the cells co-expressing CFP-Parkin and YFP, but in only about 10% of the cells co-expressing CFP-Parkin and YFP-Bcl-xL (Fig. 2D), suggesting that Bcl-xL inhibited PINK1/Parkin-dependent mitophagy. Western blotting analysis was used to further testify whether Bcl-xL inhibited the loss of mitochondrial proteins. Before treatment with CCCP, YFP-Parkin was transfected into cells alone or together with Bcl-xL for 24 hr. Western blotting analysis showed that CCCP treatment for 24 hr did not induce reduction of the mitochondrial proteins TOM20 and Smac in the cells without transfection with Parkin, but induced significant reduction of mitochondrial proteins TOM20 and Smac in the cells expressing Parkin, which was prevented by co-expressing of Bcl-xL (Fig. 2E), further indicating that Bcl-xL inhibited PINK1/Parkin-dependent mitophagy. 3.3 Bcl-xL prevents mitochondrial distribution of Parkin

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To inspect the influence of Bcl-xL on the mitochondrial distribution of Parkin, cells were transiently co-transfected with YFP-Parkin together with CFP or CFP-Bcl-xL. After CCCP treatment for 6 hr, Mitotracker deep Red was used to label mitochondria before fluorescence imaging. In contrast to the mitochondrial YFP-Parkin accumulation in the cells co-expressing YFP-Parkin and CFP, the cells co-expressing YFP-Parkin and CFP-Bcl-xL exhibited even distribution of Parkin (Fig. 3A), verifying that mitochondrial distribution of Parkin was antagonized by Bcl-xL. In order to further verify the inhibition of Bcl-xL on CCCP-induced mitochondrial translocation of Parkin, we assessed the impact of Bad on the inhibitory effect of Bcl-xL on mitochondrial Parkin accumulation by using Bad-P2A-CFP-Bcl-xL that enables Bad and CFP-Bcl-xL equimolar co-expression (using a 2A self-cleaving peptide sequence) (Lopez et al., 2016; Szymczak et al., 2004). Bad and Bcl-xL co-expressed in the cells transfected with Bad-P2A-CFP-Bcl-xL vector were verified by Western blot analysis (Yang et al., 2019). As shown in Figure 3A, in the cells co-expressing YFP-Parkin and Bad-P2A-CFP-Bcl-xL, CCCP treatment for 6 hr induced mitochondrial Parkin distribution, similar to the cells co-expressing YFP-Parkin and CFP, indicating that Bad disturbed the inhibitory role of Bcl-xL in cytosolic Parkin translocation to mitochondria. Statistical results from at least 100 cells showed that about 55% of the cells co-expressing YFP-Parkin or YFP-Parkin and CFP exhibited mitochondrial Parkin accumulation, but only 15% of the cells

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co-expressing YFP-Parkin and CFP-Bcl-xL exhibited mitochondrial Parkin translocation, further verifying the notion that Bcl-xL attenuated the translocation of Parkin from cytoplasm to mitochondrial. Based on the fact that Bcl-xL effectively restrained the accumulation of mitochondrial Parkin (Figures 3A and B), we used fluorescence loss in photobleaching (FLIP) analyses to further verify this issue. After the cells transfected with YFP-Parkin or co-transfected YFP-Parkin and CFP-Bcl-xL were treated with CCCP for 6 hr, a region in nuclear was chosen to repeatedly bleach by the maximal 514 nm laser line (Figure 3C, white square) and subsequently imaged CFP and YFP after per bleach as described previously (Edlich et al., 2011; Wang, B. et al., 2019). A ROI (Region of interest) of an adjacent cell was monitored for cell-specific bleaching. As shown in Figure 3C, after 6 hr CCCP treatment, the mitochondrial YFP-Parkin fluorescence in the cells expressing YFP-Parkin alone did not decay during repeated bleaching in nuclear region, whereas the mitochondrial YFP-Parkin fluorescence, same as the cytosolic YFP-Parkin fluorescence, of the cells in the presence of CFP-BCL-xL rapidly weakened during the repeated bleaching in nuclear region, further indicating the even distribution of Parkin in the cells co-expressing YFP-Parkin and CFP-Bcl-xL. Figure 3D shows the fluorescence intensity curves of YFP-Parkin on mitochondria or cytoplasm from 15 ROI in FLIP experiment, which is fitted by one Exponential Decay model (Fig. 3D), further proving that Bcl-xL restrained mitochondrial distribution of Parkin instead of its retranslocation from mitochondria to cytoplasm.

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3.4 Bcl-xL physically interacts with Parkin in cytoplasm

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To examine the Bcl-xL-Parkin interaction in cytoplasm, we performed quantitative E-FRET measurements to obtain the ED and RC between CFP-Bcl-xL and YFP-Parkin in living cells. Figures 3A and B exhibit the fluorescence images of typical cells and the corresponding ED and RC images. The average ED value in the RC range from 1 to 2 was counted from about 35 cells. The average ED value of cells co-expressing CFP-Bcl-xL and YFP-Parkin was 0.037 ± 0.018, higher than the 0.032 ± 0.001 for the average ED value of cells co-expressing CFP and YFP (Fig. 3C), suggesting that Bcl-xL physically interacted with Parkin in cytoplasm to restrain the mitochondrial translocation of Parkin. 3.5 Bcl-xL physically interacts with PINK1 on mitochondria We tested the influence of Bcl-xL on the location of PINK1. Consistent with previously reported results (Beilina et al., 2005; Takatori et al., 2008), in the cells expressing YFP-PINK1 alone, YFP-PINK1 was present in cytoplasm in the control cells but on mitochondria in the CCCP-treated cells (Fig. 5A; first and second row). Co-expression of Bcl-xL did not affect mitochondrial YFP-PINK1 localization with CCCP treatment for 3 hr (Fig. 5A; third and fourth row). Statistical results showed that CCCP treatment markedly increased mitochondrial PINK1distribution from 10%

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to 50% in the cells expressing YFP-PINK1, which was not restrained by co-expression of Bcl-xL (Fig. 5B), indicating that Bcl-xL did not inhibit CCCP-induced mitochondrial localization of YFP-PINK1. To examine whether Bcl-xL physically interacts with PINK1 on mitochondria, we performed quantitative E-FRET imaging for the cells co-expressing CFP-Bcl-xL and YFP-PINK1, and the cells co-expressing CFP-Bcl-xL and YFP-ActA were used as control. Figures 5C and D exhibit the three channels images of typical cells and the corresponding ED and RC images. The average ED value in the RC range from 1 to 2 was calculated from about 35 cells co-expressing CFP-Bcl-xL and YFP-PINK1 or co-expressing CFP-Bcl-xL and YFP-ActA. The average ED value of cells co-expressing CFP-Bcl-xL and YFP-PINK1 was 0.136 ± 0.010, much higher than the ED value of cells co-expressing CFP-Bcl-xL and YFP-ActA (Fig. 5F), suggesting that Bcl-xL physically interacted with PINK1. Based on the fact that Bcl-xL is preferentially bound by Bad (Yang et al., 2019), we used the Bad-P2A-CFP-Bcl-xL and YFP-PINK1 to co-transfect cells for quantitative E-FRET measurements, and Figure 5E exhibits the three channels images of typical cells and the corresponding ED and RC images. The average ED value in the RC range from 1 to 2 was calculated from about 35 cells co-expressing Bad-P2A-CFP-Bcl-xL and YFP-PINK1 was 0.037 ± 0.018, consistent with that between CFP-Bcl-xL and YFP-ActA (Fig. 5F), further verifying that Bad prevented the binding of Bcl-xL to PINK, and also demonstrating that Bcl-xL directly interacted with PINK1. In order to verify the interactions between endogenous Bcl-xL and PINK1/Parkin, co-immunoprecipitation assays were performed in HEK293T cells (Fig. 5G). As shown in Figure 5G, endogenous Bcl-xL interacted with endogenous PINK1 and Parkin, which was slightly enhanced by CCCP treatment, further verifying the direct interaction between Bcl-xL and PINK1 /Parkin.

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Discussion

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The inhibitory role of Bcl-xL in PINK1/Parkin-dependent mitophagy is a vigorously debated topic (Arena et al., 2013; Hollville et al., 2014). In over-expression model system, our data support the view that Bcl-xL inhibits mitophagy by physically interacting with both PINK1 and Parkin. Co-immunoprecipitation analyses on endogenous Bcl-xL, PINK1 and Parkin in HEK293T cells verified their direct interaction, which is consistent with previous researches (Arena et al., 2013; Hertz et al., 2013; Hollville et al., 2014). Of the utmost importance, we here also uesd live-cell imaging techniques to firmly demosntrate that Bcl-xL does not retrotranslocate Parkin from mitochondria to cytoplasm, and Bcl-xL physically interacts with Parkin in cytoplasm and with PINK1 on mitochondria to prevent mitochondrial Parkin accumulation. Live-cell imaging and FLIP analyses demonstrate that Parkin is even distribution in cells over-expressing Bcl-xL under CCCP treatment. Live-cell quantitative FRET analyses verify that Bcl-xL directly interacts with PINK1 and Parkin.

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In concrast to the speculation that Bcl-xL inhibits PINK1/Parkin-dependent mitophagy via retrotranslocating Parkin from mitochondria to cytoplasm (Hollville et al., 2014), our data (Figs. 4 and 5) surpport the view that Bcl-xL directly interacts with PINK1 and Parkin to prevent mitochondrial accumulation of Parkin. Live-cell imaging and FLIP analyses demonstrated that Parkin was even distribution in the cells with Bcl-xL over-expression (Fig. 3). The higher ED value (0.072 ± 0.003) between CFP-Bcl-xL and YFP-Parkin than that between CFP and YFP (0.032 ± 0.001) (Figs. 4A-C) suggest that Bcl-xL directly interacts with Parkin in cytoplasm, which may prevent the mitochondrial accumulation of Parkin. It was reported that Parkin translocated to depolarized mitochondria dependent on PINK1 (Narendra et al., 2008; Vives-Bauza et al., 2010). Bcl-xL is located in both cytoplasm and mitochondria, while PINK1 is stably located on depolarized mitochondria (Fig. 5A) (Fedorowicz et al., 2014; Narendra, D.P. et al., 2010; Okatsu et al., 2015; Okatsu et al., 2012). The higher ED value (0.136 ± 0.010) between CFP-Bcl-xL and YFP-PINK1 than that (0.055 ± 0.015) between CFP-Bcl-xL and YFP-ActA (Figs. 5C and D) demonstrated the interaction between Bcl-xL and PINK1 in mitochondria. Many evidences (Arena et al., 2013; Hollville et al., 2014) have indicated that Bcl-xL interacts with PINK1 and Parkin, but our live-cell quantitative FRET analyses further demonstrate that Bcl-xL interacts with PINK1 on mitochondria and with Parkin in cytoplasm. Our data approve the view that Bad disrupts the interaction between Bcl-xL and Parkin or PINK1 (Figs. 3A and 5). Based on our recent finding on the high affinity between Bcl-xL and Bad (Yang et al., 2019) and previous reports that Bcl-xL has the ability to counteract the translocation of Parkin to depolarized mitochondria, over-expression of Bad can accelerate the mitochondrial translocation of Parkin whereas knokdown of Bad fails to modulate Parkin mitochondrial translocation (Hollville et al., 2014), we conjectured that Bad regulates the translocation of Parkin by binding to endogenous Bcl-xL. Associated with our finding that in contrast to the even distribution of Parkin in the cells co-expressing CFP-Bcl-xL and YFP-Parkin, Parkin was accumulation on mitochondria in the cells co-expressing Bad-P2A-CFP-Bcl-xL and YFP-Parkin (Fig. 3A), we qualitatively speculated that Bcl-xL and Bad exhibited the higher affinity than that between Bcl-xL and PINK1. In the presence of equimolar expression of Bad and Bcl-xL, the ED value between CFP-Bcl-xL and YFP-PINK1 was less than 0.04, further demonstrating that Bad can disturb the binding of Bcl-xL and PINK1 (Figs. 5D-E). In addition, Bcl-xL also bound preferentially to Bad in cytoplasm in the presence of both Parkin and Bad (Fig. 3A). It has been reported that the Bcl-xL forms a shield in the dimer by inserting the hydrophobic C-terminal of one into the hydrophobic groove of the other one (Aranovich et al., 2012; Bleicken et al., 2017; Jeong et al., 2004). The hydrophobic C-terminal tails of Bad also inserts into the hydrophobic groove of the Bcl-xL to form a shield in the dimer (Yang et al., 1995). Our data of disruption of the interaction between Bcl-xL and PINK1 or Parkin by Bad (Figs. 3A and 5) further verified the direct interaction of Bcl-xL with PINK1 or Parkin and the view that the oligomers of Bcl-xL and Bad do not bind to PINK1 or Parkin.

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The interaction between endogenous PINK1 and Bcl-xL remains controversial. Hollville and colleagues demonstrated that Bcl-xL interacted with over-expression PINK1 instead of endogenous PINK1 (Hollville et al., 2014). However, Arena and colleagues testified that Bcl-xL interacted with both over-expressing PINK1 and endogenous PINK1 (Arena et al., 2013). Our data that PINK1 was in cytoplasm in control cells but on mitochondria in the cells treated with CCCP for 2 hr and then formed dot-like structures beside to mitochondria in the cells treated with CCCP for 6 hr (Fig. S3) indicate that CCCP induced PINK1 translocation from cytoplasm to mitochondria and subsequent formation of dot-like structures beside to mitochondria. The cells treated with CCCP for 2 hr showed higher ED value between YFP-PINK1 and CFP-Bcl-xL than that between CFP-Bcl-xL and YFP-ActA in control cells (Fig. 5). However, the cells treated with CCCP for 6 hr showed the same lower ED value between YFP-PINK1 and CFP-Bcl-xL as that between CFP-Bcl-xL and YFP-ActA in the control cells (Fig. S3), further indicating that PINK1 first interacts with Bcl-xL to form heterodimer and then separates during CCCP treatment. Yamano and Youle verified that endogenous PINK1 showed an aggregate distribution pattern indistinguishable from over-expression PINK1 (Yamano and Youle, 2013). Hollville et al demonstrated that Bcl-xL did not interact with endogenous PINK1 in the cells treated with CCCP for 6 hr, which may be due to the separation of endogenous PINK1 from Bcl-xL. Therefore, our data support the view that Bcl-xL interacts with both endogenous PINK1 and over-expression PINK1. It is interesting that Bcl-xL promotes the degradation of Parkin (Fig. S2). In most cancer cells, Bcl-xL is frequently overexpressed (Sasi et al., 2009; Walensky, 2006) and Parkin is frequently deleted (Gong et al., 2014). It has been reported that loss of Parkin results in the accumulation of Bcl-xL (Gong et al., 2017). Due to the low expression of Parkin in Hela cells, we here transfected Parkin plasmid into Hela cells. Western blotting analysis exhibited that the level of Parkin in the cells co-transfected with Parkin and Bcl-xL were significantly lower than that in the cells transfected with Parkin alone, which could be rescued by proteasome inhibitor MG 132 (Fig. S2), demonstrating that co-expression of Bcl-xL promotes the degradation of Parkin. The degradation of Parkin by Bcl-xL may contribute to the inhibitory role of Bcl-xL in PINK1/Parkin-dependent mitophagy. Parkin controls the degradation of Bcl-xL by the ubiquitin proteasome system (Gong et al., 2017), but the mechanism of Bcl-xL promoting the degradation of Parkin is unclear. According to our data, we show a reasonable mechanism by which Bcl-xL prevents mitochondrial accumulation of Parkin in Figure 6. Upon the dissipation of ΔΨm induced by CCCP, Parkin is recruited to mitochondria by PINK1 through an unclear mechanism - an initial step triggering successive events in mitophagy (Shiba-Fukushima et al., 2012). Bcl-xL prevents the mitochondrial recruitment of Parkin through two regulation ways: Bcl-xL and Parkin competitively bind with PINK1 on mitochondria to inhibit its’ recruitment capacity on Parkin; Bcl-xL physically sequestrates Parkin in cytoplasm to prevent the binding of Parkin and PINK1. Our work provides a new insight about how Bcl-xL inhibits PINK1/Parkin-dependent mitophagy.

Funding This work was supported by the National Natural Science Foundation of China (grant numbers: 61527825, 61875056 and 81572184).

Author contributions

Conflict of interest statement

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The authors declare that no competing interests exist.

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SY, TSC and XPW conceived the study, designed the experiments and wrote the manuscript. SY and MYD performed experiments. SY, MYD, AY, ZHM, YW, MXZ, XPW and TSC analyzed and interpreted the data. All authors discussed, read and approved the final manuscript.

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Acknowledgments

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We thank Dr. Andrew P. Gilmore for providing CFP-BCL-XL plasmid and Dr. Noriyuki Matsuda for insightful comments on the time of CCCP treatment.

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Fig. 1. CCCP induces PINK1/Parkin-dependent mitophagy. (A) Fluorescence images of cells expressing CFP-Parkin or CFP. The cells were treated with CCCP (10 μM) for 0 hr, 6 hr and 24 hr, respectively. Mitochondria were labeled with TOM20 by immunostaining. The TOM20 (-) cells were outlined in dotted lines. (B) The percent of cells with mitochondrial and cytosolic distribution of CFP-Parkin counted from at least 100 cells expressing CFP-Parkin. (C) The percent of cells with (+) or without (-) the mitochondrial marker TOM20 counted from at least 100 cells expressing CFP-Parkin. (D) Fluorescence images of cells transfected with CFP-Parkin and RFP-LC3. The cells were treated with CCCP (10 μM) for 0 hr and 6 hr, respectively. Mitochondria were labeled by Mitotracker Deep Red. (E) The percent of cells with co-localization between RFP-LC3 and mitochondria counted from at least 100 CFP-Parkin-positive cells. (F) Fluorescence images of cells transfected with CFP-Parkin and scrambled (Scr) siRNA (50 nM) or siPINK1 (50 nM). The cells were treated with CCCP (10 μM) for 6 hr. Mitochondria were labeled

with TOM20 by immunostaining. (G) The percent of cells with mitochondrial and cytosolic distribution of CFP-Parkin counted from at least 100 CFP-Parkin-positive cells.

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Error bars exhibits with mean ± SD obtained from three independent experiments; Statistical significance was assessed by two-tailed paired Student’s t test; ***p ≤ 0.0001; Scale bar, 10 µm.

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Fig. 2. Bcl-xL inhibits PINK1/Parkin-dependent mitophagy. (A) Fluorescence images of cells transfected with CFP or CFP-Bcl-xL. The cells were stained with mitochondrial membrane potential dye of DiIC1(5) after treatment with CCCP (10 μM) for 0 hr or 20 hr. (B) The percent of cells with negative DiIC1(5) counted from at least 100 cells expressing CFP or CFP-Bcl-xL. (C) Microscopic images of cells transfected with CFP-Parkin constructs together with YFP or YFP-Bcl-xL constructs. The cells were immunostained with TOM20 after 24 hr CCCP (10 μM) treatment. (D) The percent of cells with TOM20 (+) or (-) counted from at least 100 cells co-expressing CFP-Parkin and YFP or YFP-Bcl-xL. (E) Level of Mitochondrial proteins TOM20 and Smac assessed by Western blotting. YFP-Parkin was transfected into cells alone or together with Bcl-xL before CCCP treatment for 24 hr. Error bars exhibits with mean ± SD obtained from three independent experiments; Statistical significance was assessed by two-tailed paired Student’s t test; NS, not significant; ***p ≤ 0.0001; Scale bar, 10 µm.

ro of -p re lP na ur Jo Fig. 3. Bcl-xL prevents mitochondrial distribution of Parkin. (A) Fluorescence images of cells expressing YFP-Parkin alone or together with CFP-Bcl-xL/Bad-P2A-CFP-Bcl-xL. The cells were stained with the dye of Mitotracker Deep Red after 6 hr CCCP (10 μM) treatment. (B) The percent of cells with mitochondrial and cytosolic CFP-Parkin distribution counted from at least 100 cells.

Error bars exhibits with mean ± SD obtained from three independent experiments. (C) FLIP analyses of YFP-Parkin in the absence or presence of CFP-Bcl-xL with CCCP treatment for 6 hr. (D) Fluorescence intensity curves of YFP-Parkin on mitochondria in the absence of CFP-Bcl-xL (red) and YFP-Parkin on mitochondria (blue) and cytoplasm (green) in the presence of CFP-Bcl-xL. Fluorescence intensity curves (black) of adjacent cell served as control. (E) FLIP analyses on the retrotranslocation of mitochondrial CFP-Bcl-xL in the absence or presence of YFP-Parkin with CCCP treatment for 6 hr. (F) Fluorescence intensity curves of CFP-Bcl-xL on mitochondria in the presence of YFP (blue) and YFP-Parkin (red). Fluorescence intensity curves (black) of adjacent cell served as control. Error bars exhibits with mean ± SEM obtained from 16 ROI (Region of interest) measurements of per condition. Statistical significance was assessed by two-tailed paired Student’s t test; NS, not significant; ***p

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≤ 0.0001; Scale bar, 10 µm.

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Fig. 4. Bcl-xL physically interacts with Parkin in cytoplasm. (A-B) FRET images of living cells co-expressing CFP and YFP or CFP-Bcl-xL and YFP-Parkin. (C) The average ED values corresponding to (A) and (B), respectively. Error bars exhibits with mean ± SD obtained from at least 35 cells; Statistical significance was assessed by two-tailed paired Student’s t test; ***p ≤ 0.0001; Scale bar, 10 µm.

ro of -p re lP na ur Jo Fig. 5. Bcl-xL physically interacts with PINK1 on mitochondria. (A) Fluorescence images of cells expressing YFP-PINK1 alone or together with CFP-Bcl-xL. The cells were immunostained with TOM20 after treatment with CCCP (10 μM) for 0 h or 3 hr. (B) The percent of cells with mitochondrial and cytosolic YFP-PINK1 distribution counted from at least 100 cells expressing YFP-PINK1 or co-expressing YFP-PINK1 and CFP-Bcl-xL. Error bars exhibits with mean ± SD

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obtained from three independent experiments. (C-E) FRET images of cells co-expressing CFP-Bcl-xL and YFP-ActA, CFP-Bcl-xL and YFP-PINK1 and Bad-P2A-CFP-Bcl-xL and YFP-PINK1. (F) The average ED values corresponding to (C), (D) and (E), respectively. Error bars exhibits with mean ± SD obtained from at least 35 cells; (G) Immunoprecipitation assays on endogenous Bcl-xL, PINK1 and Parkin in HEK293T cells. Lysates prepared from HEK293T cells with CCCP (50 μM) treatment for 0 hr or 3 hr were subjected to immunoprecipitation with anti-Bcl-xL antibody, followed by anti-PINK1 and anti-Parkin immunoblotting. Statistical significance was assessed by two-tailed paired Student’s t test; NS, not significant; ***p ≤ 0.0001; Scale bar, 10 µm.

Fig.6. Schematic model showing the regulation mechanism by which Bcl-xL prevents

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mitochondrial Parkin accumulation.