Miro proteins prime mitochondria for Parkin translocation and mitophagy
2018; Springer Nature; Volume: 38; Issue: 2 Linguagem: Inglês
10.15252/embj.201899384
ISSN1460-2075
AutoresDzhamilja Safiulina, Malle Kuum, Vinay Choubey, Nana Gogichaishvili, Joanna Liiv, Miriam A. Hickey, Michal Cagalinec, Merle Mandel, Akbar Zeb, Mailis Liiv, Allen Kaasik,
Tópico(s)Mitochondrial Function and Pathology
ResumoArticle30 November 2018Open Access Source DataTransparent process Miro proteins prime mitochondria for Parkin translocation and mitophagy Dzhamilja Safiulina Corresponding Author Dzhamilja Safiulina [email protected] orcid.org/0000-0002-4188-4207 Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Malle Kuum Malle Kuum orcid.org/0000-0002-4681-1859 Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Vinay Choubey Vinay Choubey Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Nana Gogichaishvili Nana Gogichaishvili Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Joanna Liiv Joanna Liiv Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Miriam A Hickey Miriam A Hickey Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Michal Cagalinec Michal Cagalinec Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Merle Mandel Merle Mandel Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Akbar Zeb Akbar Zeb Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Mailis Liiv Mailis Liiv Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Allen Kaasik Corresponding Author Allen Kaasik [email protected] orcid.org/0000-0002-4850-3198 Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Dzhamilja Safiulina Corresponding Author Dzhamilja Safiulina [email protected] orcid.org/0000-0002-4188-4207 Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Malle Kuum Malle Kuum orcid.org/0000-0002-4681-1859 Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Vinay Choubey Vinay Choubey Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Nana Gogichaishvili Nana Gogichaishvili Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Joanna Liiv Joanna Liiv Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Miriam A Hickey Miriam A Hickey Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Michal Cagalinec Michal Cagalinec Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Merle Mandel Merle Mandel Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Akbar Zeb Akbar Zeb Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Mailis Liiv Mailis Liiv Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Allen Kaasik Corresponding Author Allen Kaasik [email protected] orcid.org/0000-0002-4850-3198 Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia Search for more papers by this author Author Information Dzhamilja Safiulina *,1,‡, Malle Kuum1,‡, Vinay Choubey1,‡, Nana Gogichaishvili1, Joanna Liiv1, Miriam A Hickey1, Michal Cagalinec1,‡, Merle Mandel1, Akbar Zeb1, Mailis Liiv1 and Allen Kaasik *,1 1Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia ‡These authors contributed equally to this work ‡Present address: Department of Cellular Cardiology, Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia *Corresponding author. Tel: +372 7374353; E-mail: [email protected] *Corresponding author. Tel: +372 7374350; E-mail: [email protected] The EMBO Journal (2019)38:e99384https://doi.org/10.15252/embj.201899384 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The Parkinson's disease-associated protein kinase PINK1 and ubiquitin ligase Parkin coordinate the ubiquitination of mitochondrial proteins, which marks mitochondria for degradation. Miro1, an atypical GTPase involved in mitochondrial trafficking, is one of the substrates tagged by Parkin after mitochondrial damage. Here, we demonstrate that a small pool of Parkin interacts with Miro1 before mitochondrial damage occurs. This interaction does not require PINK1, does not involve ubiquitination of Miro1 and also does not disturb Miro1 function. However, following mitochondrial damage and PINK1 accumulation, this initial pool of Parkin becomes activated, leading to the ubiquitination and degradation of Miro1. Knockdown of Miro proteins reduces Parkin translocation to mitochondria and suppresses mitophagic removal of mitochondria. Moreover, we demonstrate that Miro1 EF-hand domains control Miro1's ubiquitination and Parkin recruitment to damaged mitochondria, and they protect neurons from glutamate-induced mitophagy. Together, our results suggest that Miro1 functions as a calcium-sensitive docking site for Parkin on mitochondria. Synopsis Parkin translocation to damaged mitochondria is one of the earliest events that promote damage-induced mitophagy. Miro proteins function as a calcium-dependent docking site and safety switch for Parkin on healthy mitochondria, thereby controlling initiation of damage-induced mitophagy. A small pool of Parkin binds Miro1 in a PINK1-independent manner before mitochondrial damage occurs. This Parkin-Miro1 interaction does not involve Miro1 ubiquitination, nor does it affect Miro1 function. Knockdown of Miro proteins reduces Parkin translocation to mitochondria and suppresses mitophagy. Miro1 ubiquitination and Parkin recruitment to damaged mitochondria depend on calcium-binding EF-hand domains of Miro1. Introduction Maintenance of proper mitochondrial function to provide cellular energy and handle calcium is essential for cell physiology, especially for highly complex cells such as neurons. Dysfunctional or depolarised mitochondria are removed by mitophagy, the selective degradation of mitochondria, to prevent cellular damage. A drop in mitochondrial membrane potential is associated with accumulation of the serine/threonine kinase PINK1 on the outer mitochondrial membrane, which together with the ubiquitin ligase Parkin, governs the elimination of defective mitochondria by mitophagy. PINK1 phosphorylates ubiquitin, and phospho-ubiquitin serves as a mitophagy signal on mitochondria (Lazarou et al, 2015). Phospho-ubiquitin activates Parkin (Ordureau et al, 2014; Okatsu et al, 2015; Tang et al, 2017), which in turn ubiquitinates mitochondrial outer membrane proteins. There are a number of Parkin substrates on the mitochondrial outer membrane (Sarraf et al, 2013; Martinez et al, 2017). Ubiquitination of TOM20 serves as the signal for mitophagy, and overexpression of TOM20 promotes mitophagy by increasing the pool of substrates available for ubiquitination (Bingol et al, 2014). Parkin ubiquitinates VDAC1 and VDAC3 (Geisler et al, 2010; Sun et al, 2012), and silencing of VDAC1 results in significantly reduced translocation of Parkin to damaged mitochondria and prevents mitochondrial clearance (Geisler et al, 2010). Ubiquitination of Mfn2 prevents fusion of damaged mitochondria and likely leads to fragmentation as fission processes remain functional (Choubey et al, 2014). Parkin also ubiquitinates the mitochondrial outer membrane Rho GTPases Miro1/2, which are components of the adaptor complex that anchors mitochondria to motor proteins. This leads to mitochondrial arrest and may further facilitate the removal of damaged mitochondria by mitophagy (Wang et al, 2011). Miro proteins directly interact with PINK1 (Weihofen et al, 2009; Wang et al, 2011; Birsa et al, 2014), but the role of PINK1 in Miro1 degradation remains unclear. Initial findings that PINK1 phosphorylates Miro1 at serine position 156 (Wang et al, 2011) were not confirmed in later studies (Liu et al, 2012; Birsa et al, 2014; Kazlauskaite et al, 2014a). Birsa et al (2014) found that mitochondrial damage triggers prompt PINK1- and Parkin-dependent Miro1 ubiquitination, while Miro2 degradation had a much slower onset. Thus, Miro1 ubiquitination rather than its degradation has been suggested to be a signal for mitochondrial arrest. A number of recent studies have focused on Miro1 as a substrate in the PINK1/Parkin pathway, and some ubiquitination sites have been identified (Sarraf et al, 2013; Kazlauskaite et al, 2014a; Ordureau et al, 2014; Klosowiak et al, 2016). Multi-monoubiquitination of Miro1 (Kazlauskaite et al, 2014a; Klosowiak et al, 2016) and an atypical lysine-27-mediated ubiquitin chain (Birsa et al, 2014) suggest additional roles for Miro1 ubiquitination beyond degradation. Furthermore, Park et al (2017) showed recently that Miro1 enhances Parkin catalytic activity. Miro proteins have a unique structure as they have two GTPase domains and two Ca2+-binding domains—the EF-hands (Fransson et al, 2003; Frederick et al, 2004; Guo et al, 2005). Miro is involved in the regulation of mitochondrial movement by Ca2+, a function that requires Ca2+ binding to the EF-hands. This binding causes the motor/adaptor complex to dissociate from microtubules and leads to the cessation of movement (Saotome et al, 2008; MacAskill et al, 2009; Wang & Schwarz, 2009). Although the role of Ca2+ signalling in mitochondrial function is well defined, the involvement of Ca2+ in mitophagy is still poorly understood (East & Campanella, 2013; Rimessi et al, 2013). In neurons, glutamate-induced stress, which is tightly associated with Ca2+ overload, causes Parkin translocation to mitochondria (Van Laar et al, 2015). Inhibition of Ca2+ entry by voltage-gated L-type Ca2+ channel blockers or Ca2+ chelation prevents mitochondrial degradation (Cherra et al, 2013) and decreases PINK1 expression and mitophagy (Gómez-Sánchez et al, 2014). Thus, cessation of mitochondrial motility in response to elevated Ca2+ could be involved in the initial steps of mitophagy. We hypothesised that Miro1 is not only a substrate for PINK1/Parkin-dependent degradation but might also have additional roles in the regulation of mitophagy. Here, we demonstrate that Miro proteins can also function as a calcium-dependent docking site and safety switch for Parkin recruitment. Results Silencing of Miro1 and Miro2 suppresses Parkin translocation to mitochondria We used specific shRNAs targeted against Miro1 and Miro2 isoforms, to test whether these isoforms are required for the Parkin translocation to mitochondria. These shRNAs reduced effectively Miro1 and Miro2 expression at the mRNA level (Fig EV1A) and when combined, they almost completely inhibited mitochondrial mobility in primary cortical neurons (median mitochondrial velocity in the Miro1 and Miro2 shRNA-treated group was 4.3% of control group value, P < 0.0001). Click here to expand this figure. Figure EV1. Miro shRNAs specifically suppress the expression of Miro1 and Miro2 and do not affect the expression level of overexpressed PINK1 PC6 cells were transfected with either scrambled shRNA, Miro1 shRNA or Miro2 shRNA and selected for 7 days with 200 μg/ml G418. Total RNA was extracted using RNAeasy mini kit (Qiagen), and first-strand synthesis was performed using 5 μg of total RNA with Maxima First Strand cDNA Synthesis Kit (Thermo). cDNAs were subjected to qPCR using specific primers for CYC (housekeeping gene), Miro1 and Miro2 using a QuantStudio 12K Flex from Applied Biosystems by Life technologies. Acquired data were analysed using the delta Ct method and normalised to transfection efficiency, which was estimated separately. *P < 0.05 and ****P < 0.0001 compared with respective scrambled shRNA group, n = 3 samples, one-way ANOVA. Representative Western blot image of untagged PINK1 expression in PC6 cells expressing either scrambled shRNA or a combination of Miro1 and Miro2 shRNA. Note that the level of endogenous Miro proteins detected with Miro antibody recognising both isoforms was clearly decreased in the Miro shRNA-treated samples. Quantification of PINK1 band intensity from six independent samples shows that there is no statistical significance between the groups (t-test with Welch's correction). Treatment with A&O (both 15 μM) for 3 h led to clearly visible endogenous PINK1 accumulation. Data information: Data are presented as means ± SEM. Source data are available online for this figure. Download figure Download PowerPoint We tested whether Miro proteins participate in PINK1-induced Parkin translocation to mitochondria. PC6 cells were transfected with fluorescent EYFP-Parkin, scrambled shRNA, or Miro1 and/or Miro2 shRNAs with or without untagged PINK1. PINK1 induced EYFP-Parkin translocation in approximately 20–30% of scrambled shRNA-expressing cells but in a significantly smaller proportion of Miro shRNA-expressing cells (Fig 1A and B). This cannot be explained by altered PINK1 levels because the latter was not affected in the Miro shRNAs-treated groups (Fig EV1B and C). Figure 1. Miro1 and Miro2 are required for Parkin translocation to mitochondria and mitophagy Representative pseudocolour images of PC6 cells transfected with EYFP-Parkin (green) and mitochondrially targeted CFP (red) with and without PINK1 and Miro1 shRNA and Miro2 shRNA. Zoomed images show translocation of EYFP-Parkin to mitochondria in PINK1-overexpressing PC6 cells. Quantification of EYFP-Parkin translocation to mitochondria. The percentage of PC6 cells with EYFP-Parkin translocated to mitochondria in response to PINK1 overexpression is lower in the Miro shRNAs-expressing groups when compared with the scrambled shRNA-expressing group (scr shRNA). ****P < 0.0001, n = 6 dishes per group, 20 fields per dish, one-way ANOVA. The kinetics of EYFP-Parkin translocation is different in PC6 cells transfected with scrambled or Miro shRNAs. Cells were plated in separate compartments of the same dish, enabling us to visualise Parkin translocation simultaneously over 3 h under similar conditions in response to antimycin with oligomycin treatment (A&O, both 10 μM). The spatial heterogeneity of EYFP-Parkin (coefficient of variation of the intensity of individual pixels) was estimated for individual cells for each time point (n = 39-7 cells; P < 0.0001 difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM). Miro shRNAs mitigate the effect of A&O on EYFP-Parkin translocation. PC6 cells transfected with EYFP-Parkin with and without Miro shRNAs were treated with DMSO or A&O (both 10 μM) for 3 h. The figure demonstrates that the percentage of cells with Parkin translocated to mitochondria was lower in the Miro shRNAs-expressing group when compared with the scrambled shRNA-expressing group. ****P < 0.0001, n = 4 dishes, 20 fields per dish, one-way ANOVA. Miro shRNAs inhibit PINK1-induced mitophagy in PC6 cells. The figure shows the changes in the excitation spectra of mitochondrially targeted pH-sensitive Keima (ratio of 561 nm/458 nm fluorescence intensities). ****P < 0.0001, n = 80 cells from 4 dishes, Kruskal–Wallis test. Miro shRNAs inhibit basal mitophagy in primary cortical neurons expressing mitochondrially targeted Keima. Mitochondria in lysosomes (acidic pH) were counted manually, by a blinded observer (**P < 0.01, n = 9 dishes, 9 fields per dish, from two independent experiments, one-way ANOVA). Miro shRNAs decrease the number of mitochondria co-localised with the autophagosome marker EGFP-LC3B in primary cortical neurons (**P < 0.01, n = 5 dishes, 9 fields per dish, one-way ANOVA). Data information: Data are presented as means ± SEM. Source data are available online for this figure. Source Data for Figure 1 [embj201899384-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint To measure the kinetics of Parkin translocation, we designed a time-lapse experiment where we followed EYFP-Parkin translocation in response to the respiratory chain inhibitor antimycin together with the ATP synthase inhibitor oligomycin in PC6 cells transfected with scrambled or Miro shRNAs. Cells were plated in separated compartments of the same dish allowing us to visualise Parkin translocation simultaneously and to avoid dish-to-dish variation. We then estimated the spatial heterogeneity of EYFP-Parkin fluorescence (by estimating the coefficient of variation of the intensity of pixels) in individual cells at different time points, to follow the ratio of mitochondrial-to-cytosolic Parkin. Further statistical analysis of these data (39–47 cells from each group) demonstrated that Miro shRNAs reduced Parkin translocation significantly (Fig 1C) with the number of cells showing Parkin translocation being significantly lower in Miro shRNA-expressing cells (Fig 1D). We also estimated mitophagy using the mitochondrially targeted pH-dependent protein Keima, the excitation spectrum of which shifts when mitochondria are delivered to acidic lysosomes. Similar to Parkin translocation, mitophagy was increased in PINK1-expressing PC6 cells and was suppressed when Miro shRNAs were co-expressed (Fig 1E). However, it should be noted that the overall intensity of the Keima signal was lower in Miro shRNAs-expressing PC6 cells, making the ratiometric analysis prone to artefacts. We therefore performed additional mitochondrial Keima experiments in neurons expressing both Miro shRNAs and counted the number of mitochondria in an acidic environment manually. We were not able to perform this experiment with PINK1 or antimycin with oligomycin because of their toxicity in neurons. Nevertheless, the data show an almost twofold decrease in basal mitophagy in response to Miro knockdown (Fig 1F). Miro shRNAs also led to an almost twofold decrease in the number of mitochondria co-localising with the autophagosome marker, EGFP-LC3B (Fig 1G). We also excluded the possibility that the change in mitochondrial motility associated with Miro loss has a primary role in the change in Parkin recruitment. Figure EV2A shows that overexpression of Syntaphilin, which inhibits mitochondrial motility similar to Miro shRNAs (median mitochondrial velocity in the Syntaphilin-overexpressing group was 5.0% of control group value, Fig EV2B), neither induced Parkin translocation nor affected Parkin translocation in the presence of PINK1 or antimycin and oligomycin (A&O). This suggests that inhibition of mitochondrial movement per se cannot be the primary reason for the altered Parkin translocation. Click here to expand this figure. Figure EV2. Syntaphilin overexpression does not affect Parkin translocation Syntaphilin overexpression has no effect on A&O- or Pink1-induced EYFP-Parkin translocation in PC6 cells (****P < 0.0001, n = 4–8 dishes, 20 fields per dish, one-way ANOVA). Syntaphilin overexpression led to an almost complete inhibition of mitochondrial motility in primary cortical neurons. Data are presented as a Tukey boxplot (****P < 0.0001, n = 305–339 individual mitochondria from 12 axons per group pooled from 3 individual dishes, Mann–Whitney test). Data information: Data are presented as means ± SEM or as a Tukey plot (median ± 1.5 times interquartile range). Source data are available online for this figure. Download figure Download PowerPoint Thus, these set of data allow us to conclude that Miro proteins are required for appropriate Parkin translocation and initiation of mitophagy. Miro1 recruits Parkin to polarised mitochondria When performing control experiments, we observed, to our surprise, that overexpression of myc-Miro1 changed Parkin localisation in the cytosol. However, the translocation pattern was completely different from PINK1- or A&O-induced Parkin translocation. In the case of PINK1 or A&O, a majority of cytosolic EYFP-Parkin translocated to a few strong individual puncta that did not cover the entire mitochondrial network (Fig 2A middle panels). In Miro1-overexpressing cells, however, the EYFP-Parkin inclusions were relatively dull and not as bright as in PINK1-overexpressing or A&O-treated cells (Fig 2A lower panels). Moreover, in Miro1-overexpressing cells, EYFP-Parkin overlapped with the mitochondrial network and did not show puncta-like structures as in A&O-treated cells. Figure 2. Miro1 recruits Parkin to polarised mitochondria A. The pattern of Parkin translocation caused by Miro1 overexpression is different from A&O-induced translocation. The figure illustrates puncta-like EYFP-Parkin on mitochondria in antimycin and oligomycin-treated (both 10 μM for 3 h) PC6 cells (middle panels), whereas in Miro1-overexpressing cells, EYFP-Parkin appears along rod-shaped mitochondria visualised with CFP-mito (lower panels). The merged panels present EYFP-Parkin (green) and CFP-mito (red). B. Overexpression of myc-Miro1 and untagged Miro2, but not myc-Miro2, induced EYFP-Parkin translocation to mitochondria. ****P < 0.0001 versus control group, n = 5–6 dishes, 20 fields per dish, one-way ANOVA or t-test. C. Quantification of EYFP-Parkin signal heterogeneity in PC6 cells demonstrates that Miro1 overexpression induces Parkin translocation from the cytosol to mitochondria, which is significantly weaker than PINK1 overexpression- or A&O (3 h)-induced translocation. **P < 0.01 and ****P < 0.0001 versus control group, n = 18–32 cells from 3 dishes per group, Kruskal–Wallis test. D, E. Miro1 overexpression does not cause mitochondrial depolarisation. (D) Representative image of Miro1 and EYFP-Parkin-transfected cells stained with TMRE. The merged panels present EYFP-Parkin (green) and TMRE (red). (E) Relative TMRE intensity quantified in neurons transfected with Miro1 (detected by CFP-mito co-transfection); the control group is non-transfected cells in the same image. The relative TMRE signal was slightly increased in Miro1 overexpressing neurons but unaffected in Miro shRNAs-expressing neurons. Note that FCCP treatment (3 μM for 15 min) almost completely abolished TMRE fluorescence. ****P < 0.0001 versus control group, n = 94–186 cells from 4 dishes per group, Kruskal–Wallis test. F. Co-expression of HA-Parkin with Miro1 does not suppress mitochondrial motility in axons of cortical neurons. Overexpression of Miro1 alone increased the motion time in both directions as well as the relative mitochondrial velocity. Co-transfection of Parkin did not affect significantly the motility parameters. Data are presented as Tukey boxplot. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, n = 689–846 individual mitochondria from 39 to 44 axons per group pooled from at least from 10 individual dishes from 4 independent experiments, Kruskal–Wallis test. H. Parkin overexpression does not induce degradation of myc-Miro1 in control conditions. Representative Western blot image and analysis of myc-Miro1 expression in HEK cells. Treatment with A&O (both 15 μM) for 3 h in the absence of MG132 led to slight degradation of myc-Miro1. Co-expression of EYFP-Parkin did not induce degradation of Miro1 although an additional band above the Miro1 band was present. However, Parkin co-expression enhanced Miro1 degradation when cells were treated with A&O. *P < 0.05 and ****P < 0.0001, ns: not significant, n = 4 independent experiments, one-way ANOVA. Note that only the main band of myc-Miro1 at 80 kDa was analysed and that YFP was used to compensate in groups not expressing EYFP-Parkin. I. EYFP-Parkin co-immunoprecipitates with overexpressed myc-Miro1, both in DMSO- and in A&O (both 15 μM for 3 h)-treated HEK cells. J. Endogenous Parkin co-immunoprecipitates with endogenous Miro1. Miro1 was immunoprecipitated from non-transfected and non-treated HEK cells using mouse monoclonal anti-Miro1 antibody and immunoblotted for endogenous Parkin. Data information: Data are presented as means ± SEM or as a Tukey plot (median ± 1.5 times interquartile range). Source data are available online for this figure. Source Data for Figure 2 [embj201899384-sup-0004-SDataFig2.xlsx] Download figure Download PowerPoint The results depicted in Fig 2B (left panel) show that the percentage of cells in which Parkin translocated to mitochondria in Miro1-overexpressing groups was similar to PINK1 overexpression (Fig 1B) or A&O treatment (Fig 1D). However, this assay does not show the strength of Parkin translocation, i.e., the amount lost from the diffuse cytosolic pool. On the other hand, the heterogeneity assay depicted in Fig 2C does show that the strength of Parkin translocation from cytosol to mitochondria within single Miro1-expressing cells is weaker when compared with PINK1-overexpressing or A&O-treated cells. Thus, Miro1 induces Parkin translocation in a similar proportion of cells to other treatments but the translocation in individual cells is significantly weaker. Miro1-induced Parkin translocation was not limited to PC6 cells as Miro1 overexpression induced similar effects in MEFs and primary cortical neurons (Appendix Fig S1). We next tested whether we could also induce Parkin translocation by overexpressing Miro2. However, overexpression of myc-Miro2 had no effect on Parkin localisation (Fig 2B, left panel) when compared with myc-Miro1 (same plasmid backbones; Fransson et al, 2003). This could be, however, explained by cellular mislocalisation of myc-Miro2, which remained largely cytosolic, while overexpressed myc-Miro1 was perfectly co-localised with a mitochondrial marker (Appendix Fig S2). We therefore repeated this experiment using untagged Miro2 that localised clearly to mitochondria and induced strong Parkin translocation (Fig 2B, right panel). These results suggest that Miro1 and Miro2 act redundantly in this case. Miro1 overexpression did not induce mitochondrial depolarisation but instead led to slight hyperpolarisation (Fig 2D and E) consistent with a previous report in Drosophila (Babic et al, 2015). Notably, Miro1 overexpression-induced Parkin translocation did not induce significant degradation of Miro1 in HEK cells at control conditions but only when the cells were treated with A&O (Fig 2G). Also, co-expression of Parkin and Miro1 did not inhibit mitochondrial trafficking (Fig 2F, Appendix Fig S3), as we initially expected (as previously reported by Wang et al, 2011), suggesting that Miro has remained functional. These microscopy findings were supported by co-immunoprecipitation experiments. Figure 2H shows that Parkin co-immunoprecipitates with Miro1 under basal conditions, i.e., without applying mitochondrial uncouplers or inhibitors of the respiratory chain. This interaction became stronger when cells were treated with A&O. In the latter case, we noted a number of higher molecular weight bands positioned approximately 9 and 20 kDa above Miro1, suggesting Miro1 (poly)ubiquitination. Furthermore, we show that the Miro1-Parkin interaction can be detected between endogenous proteins (Fig 2I). Note that we also observed interaction between overexpressed untagged Miro2 and Parkin (Appendix Fig S4). Thus, these data suggest that Miro1 is able to attract cytosolic Parkin to mitochondria without affecting the mitochondrial membrane potential or its own degradation. Miro1 recruits Parkin independently of PINK1 and of Parkin E3 ubiquitin ligase activity To test whether PINK1 is required for Miro1-induced Parkin translocation to mitochondria, we first suppressed endogenous PINK1 using specific shRNA (Choubey et al, 2014). Miro1 overexpression also induced Parkin translocation in PC6 cells transfected with PINK1 shRNA, whereas no Parkin translocation was observed when PINK1 shRNA-expressing cells were treated with the A&O (Fig 3A; note that the graphs depict the percentage of cells showing translocation not the strength of translocation itself: Miro1-induced Parkin translocation was relatively weak whereas in A&O-treated groups it was very strong). To exclude the possibility that the low remaining level of PINK1 after shRNA silencing was sufficient for Miro1-induced Parkin translocation, we repeated
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