RESEARCH ARTICLE

Identification of Miro1 and Miro2 as mitochondrial receptors formyosin XIXStefanie J. Oeding*, Katarzyna Majstrowicz*, Xiao-Ping Hu*, Vera Schwarz*, Angelika Freitag‡, Ulrike Honnert,Petra Nikolaus and Martin Bahler§

ABSTRACTMitochondrial distribution in cells is critical for cellular function andproper inheritance during cell division. In mammalian cells,mitochondria are transported predominantly along microtubules bykinesin and dynein motors that bind indirectly via TRAK1 and TRAK2 toouter mitochondrial membrane proteins Miro1 and Miro2 (Miro1/2).Here, usingproximity labelling,we identifiedMiro1/2as potential bindingpartners of myosin XIX (Myo19). Interaction studies show that Miro1binds directly to a C-terminal fragment of the Myo19 tail region and thatMiro1/2 recruit theMyo19 tail in vivo. This recruitment is regulated by thenucleotide state of the N-terminal Rho-like GTPase domain of Miro1/2.Notably, Myo19 protein stability in cells depends on its association withMiro1/2. Downregulation of Miro1/2 or overexpression of the adaptorproteins TRAK1 and TRAK2 caused a reduction in Myo19 proteinlevels.Myo19 regulates the subcellular distribution of mitochondria, anddownregulation, as well as overexpression, of Myo19 inducedperinuclear collapse of mitochondria, phenocopying loss of thekinesin KIF5, dynein or their mitochondrial receptors Miro1/2. Theseresults suggest that Miro1 and Miro2 coordinate microtubule- andactin-based mitochondrial movement.

This article has an associated First Person interviewwith the first authorof the paper.

KEY WORDS: Miro GTPase, Mitochondria, Myosin

INTRODUCTIONMitochondria are dynamic organelles that can fuse with eachother, divide, move along cytoskeletal tracks and make functionalcontacts with other membranous compartments (Friedman andNunnari, 2014). Interfering with this dynamic behaviour impairsmitochondrial function and may lead to a multitude of mostlydegenerative diseases (Nunnari and Suomalainen, 2012; Mishra andChan, 2014). In cells, mitochondria are constantly transported tosites where they are needed. This transport occurs either along actinfilaments or microtubules depending on the organism. In animalcells, mitochondria are predominantly hauled bidirectionally alongmicrotubules. The microtubule-based motor kinesin-1 (KIF5)transports mitochondria towards the plus-end of microtubules,which usually points towards the cell periphery (Tanaka et al.,1998). KIF5 is recruited to mitochondria with the help of adaptor

proteins (Stowers et al., 2002), which mediate the binding to theouter mitochondrial membrane protein Miro (Miro1 and Miro2 invertebrates; denoted Miro1/2). Miro encompasses a C-terminaltransmembrane domain and two EF-hand or ELM domains that arein turn flanked by two GTPase domains (Fransson et al., 2003;Klosowiak et al., 2013; see also Fig. S1). The N-terminal GTPasedomain shares similarity with the Rho-subfamily of monomericGTPases. An adaptor protein that links KIF5 with Miro was firstidentified in Drosophila (Glater et al., 2006). Subsequently, twomammalian homologues, TRAK1 (OIP106) and TRAK2 (GRIF-1),were identified as adaptors and shown to mediate bidirectionalmicrotubule-dependent transport of mitochondria (Brickley et al.,2005; Fransson et al., 2006; MacAskill et al., 2009a; Koutsopouloset al., 2010; Brickley and Stephenson, 2011; van Spronsen et al.,2013). Transport of mitochondria towards the microtubule minus-ends is mediated by the dynein-dynactin complex. This complexsimilarly interacts with the adaptors TRAK1 and TRAK2(van Spronsen et al., 2013; Gama et al., 2017). The microtubule-dependent transport of mitochondria can be arrested by the bindingof Ca2+ to the EF-hands of Miro (Saotome et al., 2008; MacAskillet al., 2009b; Cai and Sheng, 2009; Wang and Schwarz, 2009). Apoint mutation in the N-terminal GTPase domain of Miro that ispredicted to favour the GDP-bound state, revealed that this domainis essential for mitochondrial transport and morphology (Franssonet al., 2003; Babic et al., 2015). However, it is currently not knownwhether this GTPase domain cycles between different nucleotide-bound states under physiological conditions. The protein Alex3 ofthe Armcx gene family also regulates mitochondrial movements, inneurons, by interacting with the Miro1/2-TRAK2 complex and thisinteraction is abrogated by Ca2+ binding to the EF-hands of Miro1/2(Lopéz-Doménech et al., 2012). In addition, Miro1/2 can recruit theprotein CenpF, and thereby contribute to the mitotic redistribution ofmitochondria (Kanfer et al., 2015). The proteins Mitofusin-1/2,DISC1 andAPC are yetmore proteins that are reported to interact withthe Miro-TRAK complex (Misko et al., 2010; Norkett et al., 2016;Mills et al., 2016). The protein hypoxia upregulated mitochondrialmovement regulator (HUMMR) was shown to interact directly withMiro1/2 (Li et al., 2009). Importantly, Miro1/2 are also substrates forthe E3 ubiquitin ligase Parkin that targets them for proteasomaldegradation (Sarraf et al., 2013). Mutations in Parkin have beenassociated with Parkinson’s disease (Kitada et al., 1998).

Actin filaments in mammalian cells critically contribute tomitochondrial fission. The ER-associated formin INF2 incombination with a mitochondria-associated splice form of theactin nucleator Spire and the filament-forming motor myosin II areinvolved in constricting mitochondria to initiate their fission(Korobova et al., 2013, 2014; Manor et al., 2015; Ji et al., 2015).The myosin MYO6 is recruited to damaged, ubiquitylatedmitochondria and promotes F-actin cage assembly to preventrefusion of dysfunctional mitochondria (Kruppa et al., 2018).Received 24 April 2018; Accepted 2 August 2018

Institute of Molecular Cell Biology, Westfalian Wilhelms University Munster, 48149Munster, Germany.*These authors contributed equally to this work‡Deceased

§

Author for correspondence (baehler@uni-muenster.de)

S.J.O., 0000-0002-2349-4619; X.-P.H., 0000-0002-2340-787X

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Additionally, actin filaments serve as tracks for mitochondrialmovement. The actin-basedmotormyosin XIX (Myo19) is associatedwith mitochondria and when overexpressed induces the formation ofmotile tadpole-shaped mitochondria (Quintero et al., 2009).Downregulation of Myo19 interferes with the proper partitioning ofmitochondria during mitosis, and leads to a stochastic failure in celldivision (Rohn et al., 2014). Myo19 consists of a head, light chainbinding and tail domain (see Fig. S1). In vitro studies revealed that thehead domain is a plus-end directed motor that is firmly attached toF-actin for most of the time of its chemo-mechanical cycle (Luet al., 2014). The tail domain, on the other hand, specifies itsrecruitment to mitochondria (Quintero et al., 2009). A short motif inthe tail domain (amino acids 860-890) is able to mediate binding tothe lipids of the outer mitochondrial membrane (Shneyer et al., 2016;Hawthorne et al., 2016). However, it is not known how targetingspecificity to the outer mitochondrial membrane is achieved andwhether there is any coordination between actin- and microtubule-based movements of mitochondria.

In the present study, we attempted to gain further insight into thefunctions of Myo19 by investigating the molecular mechanism(s)of its recruitment to mitochondria. Interestingly, Myo19 shares itsmitochondrial receptor with kinesin (KIF5) and dynein thatpotentially allows for a coordination of actin- and microtubule-based mitochondrial movements.

RESULTSMiro1/2 are mitochondrial receptors of Myo19To identify potential mitochondrial receptors for Myo19, weused tail proximity-dependent biotinylation (BioID) (Roux et al.,2012), since the tail region contains the mitochondrial targetinginformation and localizes to mitochondria (Quintero et al., 2009).We generated a stably transfected HeLa cell clone that expressedlow levels of the fusion protein BirA*-Myo19 tail when incubatedwith sodium butyrate (Fig. 1A,B). Biotinylated proteins wereaffinity purified by magnetic streptavidin-beads and analysed bymass spectrometry (Table S1). Thereby, we identified peptides from

Fig. 1. Identification of potential Myo19 tail binding proteins using BioID. Stably transfected myc-BirA*-Myo19 tail HeLa cells and HeLa wild-type (wt) cellswere grown in medium supplemented with biotin. Cells were either treated overnight with (+) or without (−) sodium butyrate to enhance expression of therecombinant construct. Biotinylated proteins were purified by streptavidin affinity chromatography. Lysates (L) and eluates (E) were separated by SDS-PAGE andtransferred to a PVDFmembrane for immunoblotting. (A) The protein myc-BirA*-Myo19 tail was detected by immunoblotting with anti-myc antibody (arrowhead).Stably transfected cells treated with sodium butyrate show stronger expression of the myc-BirA*-Myo19 tail construct compared with levels in untreatedtransfected cells. (B) Immunoblot analysis with peroxidase-labelled streptavidin shows strongly enhanced biotinylation of endogenous proteins after elevatedmyc-BirA*-Myo19 tail expression in cells treated with sodium butyrate (arrowhead indicates biotinylated construct). (C) Biotinylation of Miro, as a function ofmyc-BirA*-Myo19 tail expression, enhanced by sodium butyrate treatment, is shown by immunoblotting of cell lysates and eluates from streptavidin beads.The Miro antibody recognizes Miro1 and Miro2 and cross-reacts nonspecifically in the lysates with proteins running above and belowMiro1/2. Cell lysates (L) andeluates (E) from streptavidin beads arising fromwt and stably transfected HeLa cells were loaded on the same gel, but not in adjacent lanes (splices are indicated).(D) The outer mitochondrial membrane porin VDAC-1 is not biotinylated by myc-BirA*-Myo19 tail.

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Miro1 and Miro2 (Miro1/2), the mitochondrial receptors formicrotubule-based transport. Immunoblotting experiments furtherdemonstrated that Miro1/2, but not the outer mitochondrialmembrane protein VDAC-1, were biotinylated specifically byBirA*-Myo19 tail. Miro1/2 were eluted after affinity purificationfrom streptavidin exclusively upon expression of the BirA*-Myo19tail construct (Fig. 1C,D). In accordance with our results, Miro-2has recently been identified as a binding partner of Myo19 in twoindependent large-scale protein-protein interaction screens (Huttlinet al., 2015; Hein et al., 2015). Two additional outer mitochondrialmembrane proteins were discovered in the Myo19-tail BioIDscreen, namely metaxin-3 and MAVS (Table S1). Subsequently, wefocused on Miro1/2 proteins and tested whether Myo19 interactswith Miro1 directly (Fig. 2). We expressed and purifiedC-terminally His-tagged recombinant human Miro1 lacking itstransmembrane domain (Miro1-ABC, aa 1-592; Klosowiak et al.,2016) (Fig. 2A). As a potential binding partner, we expressed andpurified a FLAG-tagged C-terminal tail fragment (aa 898-970-FLAG) of human Myo19 that lacks the more N-terminally locatedlipid-binding motif (Fig. 2A). The purified Myo19 C-tail fragmentstill contained a major contaminant protein of∼70 kDa from E. coli.Pull-down experiments were performed with Miro1 adsorbed toTalon beads. The Myo19 C-tail fragment was eluted specificallytogether with Miro1, but not from control beads lacking Miro1-ABC (Fig. 2B), indicating that this Myo19 tail fragment interactsdirectly with Miro1.To test for potential involvement of Miro1/2 in the localization

of Myo19 to mitochondria, we downregulated Miro1/2 bysiRNA in HeLa cells. Notably, downregulation of Miro1/2resulted in a concomitant downregulation of Myo19 as revealedby immunoblotting (Fig. 3A-D). This drop in Myo19 protein levelwas not due to a general reduction in mitochondrial mass, asthe signal for the outer mitochondrial membrane protein VDAC-1was not affected by downregulation of Miro1/2 (Fig. 3A,B).Transfection of cells with either pools or individual siRNAsdirected against Miro1/2 led to time-dependent downregulation ofMiro1/2, with clear reduction after 48 h and 72 h (∼85% reduction)(Fig. 3A,B). Notably, downregulation of Miro1/2 was accompaniedby comparable time-dependent downregulation of Myo19 (∼70%after 72 h). The concomitant downregulation of Myo19 with Miro1/2 could be attenuated by transient overexpression of Miro2 resistantto the siRNA. The ability of Miro2 to rescue Myo19 expressiondepended on the nucleotide-state of the N-terminal GTPase domain.Introduction of a point mutation (T18N) that in analogy to otherGTPases is predicted to induce a constitutively GDP-bound ornucleotide-free state prevented the rescue of Myo19 upon transienttransfection (Fig. 3B,D). Conversely, downregulation of Myo19with siRNA did not alter the expression of Miro1/2 (Fig. 3A,C).These results strongly argue that Miro1/2 stabilize Myo19, but thatconversely, Myo19 does not influence the stability of Miro1/2.Indirect immunofluorescence labelling of Myo19 in cells treatedwith siRNA against Miro1/2 confirmed that the amounts of Myo19protein associated with mitochondria depended on the expression ofMiro1/2 (Fig. 3E). The mitochondrial signal of Myo19 was greatlyreduced in cells with downregulatedMiro1/2. To verify thatMiro1/2indeed regulate Myo19 protein stability, pulse-chase experimentswere performed with Halo-tagged Myo19. Stable cell linesexpressing Halo-tagged Myo19 were incubated with siRNAdirected against Miro1/2 and the Halo-tag of Myo19 protein waspulse-labelled with the fluorophore TMR. Labelled Myo19 decayedfaster when Miro1/2 were simultaneously downregulated (Fig. 3F).Interestingly,Myo19 protein turnover was slower when the Halo-tag

was fused to the C-terminus (Myo19-Halo; Fig. 3G). This was thecase irrespective of whether the cells were treated with non-targetingcontrol siRNA orMiro1/2 siRNA. The stable expression of the Haloconstructs reduced the levels of endogenous Myo19 so that the sumof exogenous and endogenous Myo19 amounted to the level ofendogenous Myo19 in wild-type cells. The ratio of N-terminally-tagged Halo-Myo19 to endogenous Myo19 was 1:1, whereas theratio of Myo19-Halo to endogenous Myo19 was 1:3. These datareveal that the positioning of the tag influences the life-timeof Myo19 protein and that Miro1/2 increase the life-time ofMyo19 protein.

Fig. 2. Myo19 C-tail binds directly to Miro-1 in vitro. (A) RecombinantMiro1-ABC (aa 1-592) andMyo19 C-tail (aa 898-970) were expressed inE. coliand purified as described in the Materials and Methods. The purified proteinswere separated by SDS-PAGE and stained with Coomassie Blue. Miro1-ABCand Myo19 C-tail bands are indicated by arrowheads and molecular massmarkers are indicated on the right. (B) Pull-down assay showing that Myo19C-tail binds to His-tagged Miro1-ABC directly. Purified Miro1-ABC (input) wasbound to TALON beads and incubated with purified Myo19 C-tail. Afterwashing, Miro1-ABC was eluted with an excess of imidazole. Western blotanalysis with antibodies directed against Miro and the FLAG-tag of Myo19C-tail shows coelution of Miro1-ABC and Myo19 C-tail. Input, purified Miroprotein bound to TALON beads. FT1, flow-through of Miro1-ABC afterincubation with TALON beads; FT2, flow-through of Myo19 C-tail afterincubation with TALON beads; W, Wash; Elu1+2, eluted fractions ofMiro1-ABC. As a control, beads were incubated with buffer and Myo19 C-tailonly. Molecular mass markers are indicated on the left.

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Further evidence suggesting that release of Myo19 frommitochondria promotes its degradation was provided by theobservation that stable expression of BirA*-Myo19 tail displacedendogenous Myo19 from mitochondria without a correspondingincrease of Myo19 protein in the cytosol fraction (Fig. 4A). Analysisof cell homogenates confirmed that Myo19 is degraded uponoverexpression ofMyo19 tail (Fig. S2). Additionally, overexpression

of BirA*-Myo19 tail led to a slight, although not significant,reduction of the amount of TRAK1 that was associated with purifiedmitochondria (Fig. 4). Next, we wondered whether adaptor proteinsTRAK1 and TRAK2 could compete with Myo19 for binding toMiro1/2 and hence their overexpression would induce adownregulation of Myo19. Indeed, transient transfection of eitherTRAK1 or TRAK2 led to a downregulation of Myo19 according to

Fig. 3. See next page for legend.

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the rate of transfection (Fig. 4C,D). Comparable results wereobtained when C-terminal fragments of TRAK1 (aa 396-953) orTRAK2 (aa 324-913), which harbour the Miro-binding region, butnot the KIF5- or dynactin-binding regions, were transientlyoverexpressed (Fig. 4C,D). In contrast, Miro1/2 levels wereunaltered after transient overexpression of the TRAK constructs.These results indicate that Myo19 competes with TRAK proteins forbinding to Miro1/2.The simultaneous downregulation of Myo19 with Miro1/2

precluded a meaningful analysis of potential redistribution ofMyo19 in the absence of Miro1/2. The proportion of backgroundsignal significantly increased in relation to the specific signal, sothat no meaningful quantification of the remaining mitochondriallocalization of Myo19 was possible (see Fig. 3E). Therefore, wedevised an alternative strategy to test for in vivo recruitment ofMyo19 by Miro1/2. We retargeted Miro1 to early endosomes byreplacing its C-terminal transmembrane domain with 2xFYVEdomains that mediate binding to phosphatidylinositol-3-phosphate(see schematic representation in Fig. S1). This Miro1/2 constructwas indeed targeted to early endosomes as it colocalized withfluorescently labelled transferrin that was taken up by receptor-mediated endocytosis (Fig. S3A). To monitor retargeting of theMyo19 tail that directs Myo19 to mitochondria (Quintero et al.,2009; see also Fig. 4) and to delineate the mitochondrial and Miro1/2 binding site(s) in the Myo19 tail region more precisely, weanalysed various fragments of the Myo19 tail region for Miro-dependent retargeting to early endosomes. We found that the tailregion of Myo19 localized both to mitochondria with endogenousMiro1/2 and to early endosomes with retargeted Miro1 (Fig. 5A-C),indicating that Miro1/2 are sufficient for the specific recruitment ofMyo19 tail in vivo. An N-terminal tail fragment (aa 824-897)encompassing a reported lipid-binding domain (Shneyer et al.,2016; Hawthorne et al., 2016) localized exclusively to mitochondriaand was not redirected to early endosomes (Fig. 5A-C). In contrast,the C-terminal tail fragment (aa 898-970) that bound to Miro1in vitro, colocalized with Miro1/2 that was retargeted to earlyendosomes (Fig. 5A-C). Part of this C-terminal tail fragment wasalso found in the cytosol, but it was not obviously colocalized with

mitochondria (Fig. 5A-C). These results suggest that the tail regionof Myo19 can be targeted to mitochondria by an N-terminallipid-binding motif and a C-terminal Miro-binding domain. Forrecruitment of the Myo19 tail region, a fragment of Miro1/2containing the N-terminal GTPase domain and the two ELMdomains (aa 1-408) was sufficient (Fig. 5D-F); the C-terminalGTPase domain was dispensable. Next, we examined potentialregulation of Myo19 recruitment by the nucleotide state of theN-terminal GTPase domain of Miro. Introduction of the pointmutation T18N in the N-terminal GTPase domain abrogatedrecruitment of the Myo19 tail and C-terminal tail fragment tomitochondria (Fig. 5G-I). The correlation between localization ofretargeted Miro1/2 and TOMM20 was barely affected by thedifferent Myo19 tail constructs (Fig. S2B).

Myo19 regulates the subcellular distribution ofmitochondriaIn mammalian cells, the subcellular distribution of mitochondriais regulated by microtubule-based motor proteins and theirmitochondrial receptor Miro1/2 (Maeder et al., 2014; Mishra andChan, 2014). Silencing of Miro1/2 or Kif5 protein expression hasbeen shown to induce perinuclear accumulation of mitochondria(Tanaka et al., 1998; Liu et al., 2012). To analyse whether Myo19contributes to mitochondrial distribution in cells, we either silencedor overexpressed Myo19 in HeLa cells. We specifically measuredthe perinuclear accumulation factor of mitochondria in cells, whichwe defined as the percentage of mitochondria within the perinuclearregion, divided by the fraction of the cell area occupied by thisregion. This quantification confirmed that depletion of Miro1/2 orKif5B with siRNAs causes significant perinuclear accumulationof mitochondria (Fig. 6). Interestingly, siRNA-mediated depletionof Myo19 expression also significantly shifted the localization ofmitochondria to the perinuclear region (Fig. 6). Next, we addressedwhether overexpression of Myo19 or different tail fragments wouldaffect mitochondrial distribution. We found that overexpression ofGFP-Myo19 phenocopied the effect of Myo19 downregulation(Fig. 7). This effect was independent of the actin-based motorcapacity of Myo19, since the GFP-labelled tail region of Myo19,when overexpressed, induced an even more pronounced perinuclearaccumulation of mitochondria (Fig. 7). Overexpression of theMyo19 C-tail fragment, which has been shown to interact withMiro, also induced perinuclear accumulation of mitochondria(Fig. 7). The N-tail lipid-binding fragment might still compete tosome extent for binding of endogenous Myo19 to mitochondria, asits overexpression caused a less-pronounced, but still significant,perinuclear accumulation of mitochondria (Fig. 7). To exclude thepossibility that perinuclear accumulation of mitochondria was dueto oligomerization of the fused EGFP label, the experiments wererepeated with full-length Myo19 and Myo19 tail, respectively fusedwith a Halo tag and labelled with TMR. These experimentsconfirmed that the perinuclear accumulation of mitochondriaoccurred independently of EGFP, and again showed that the tailregion of Myo19 has a stronger effect on mitochondrial distributionthan full-length Myo19 (Fig. 8). We also assessed whetheroverexpression of Myo19 or tail fragments thereof led to a changein cell size or shape. Such changes might impact the quantificationof perinuclear clustering. We found that cells overexpressing full-length Myo19 covered a smaller area, had a shorter perimeter andincreased circularity (Fig. S4). However, this effect seemed to bedependent on the motor domain ofMyo19, since none of theMyo19tail fragments caused similar changes when overexpressed (Fig. S4).In cells overexpressing EGFP-tail, for which the most pronouncedperinuclear accumulation of mitochondria had been observed, the

Fig. 3. Downregulation of Miro1/2 protein concomitantly reduces theamount of Myo19 protein. HeLa cells were transfected with an siRNA poolagainst Myo19 or cotransfected with a pool of siRNA against Miro-1 and eithera pool of siRNA against Miro2 (A,E) or a specific siRNA against Miro2 (B-D)and the downregulation of Miro and Myo19 was monitored 48 h or 72 h later byimmunoblotting with anti-Miro and anti-Myo19 antibodies. Notably, Myo19protein was downregulated concomitantly with Miro1/2, but not vice versa.Miro1/2-knockdown cells were transiently transfected with empty vector or withmyc-Miro2 or myc-Miro2 T18N vector. VDAC-1 and β-actin served as loadingcontrols for equal amounts of mitochondrial mass and total protein,respectively. The experiment was replicated several times with comparableresults. (C,D) Quantification of results shown in A and B after 72 h. Expressionlevels of Miro (C) and Myo19 (D) were quantified after the indicated treatmentsof cells as described in B from 6 (3 for rescue with Miro2 T18N) independentexperiments. Error bars represent s.e.m. ***P≤0.001, **P≤0.01, n.s., notsignificant. Re-expression of Miro2, but not Miro2 T18N, in Miro1/2-knockdowncells restoresMyo19 protein levels. (E) Immunofluorescence staining of Myo19in cells treated with non-targeting or Miro1/2 siRNAs for 48 h. Mitochondriawere labelled with Mitotracker. Scale bars: 5 μm. (F,G) Pulse-chase labelling ofHalo-Myo19 (F) or Myo19-Halo protein (G), respectively, in HeLa cells thatwere treated with either control siRNA or Miro1/2 siRNA. HeLa cells stablyexpressing Halo-taggedMyo19were incubated with fluorescent HaloTag-TMR(tetramethylrhodamine) for 30 min. After different times of chase the amount offluorescent Halo-tagged Myo19 was quantified and plotted. n=3 for Halo-Myo19 and n=2 for Myo19-Halo. Error bars represent s.e.m.

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perinuclear accumulation factors were only weakly correlated to thecell size and shape as assessed by Pearson’s correlation coefficient.The strongest correlation was found between a cell’s perinuclearaccumulation factor and its covered area or perimeter (R=0.46 and0.43, respectively). This positive correlation confirmed rather thaninvalidated the observed increased perinuclear accumulation ofmitochondria in the smaller Myo19-overexpressing cells, sinceincreased perinuclear clustering in smaller cells would actually bedetected less readily. In summary, both the downregulation ofMyo19 and the overexpression of full-length Myo19 or Myo19 tailfragments that contain the Miro-binding site induced perinuclearclustering of mitochondria.

DISCUSSIONIn this study, we identified Miro1/2 as mitochondrial receptors forMyo19 and showed that this interaction stabilizes Myo19, but notMiro1/2. We further found that both downregulation of Myo19 andoverexpression of Myo19 or Miro-binding Myo19 fragmentsphenocopy the loss of Miro1/2 and the microtubule-based motorKIF5. KIF5-dependent transport of mitochondria towards the plus-end of microtubules and dynein-dependent transport to the oppositeend are mediated by the protein TRAK1/2, which serves as

an adaptor for Miro1/2 and both KIF5 and dynactin/dynein(Glater et al., 2006; van Spronsen et al., 2013). We furtherdemonstrate that Myo19 interacts directly with Miro1/2 andcompetes with TRAK1/2 for binding. These results indicate thatMiro1/2 coordinate actin- and microtubule-based mitochondrialmovements. The perinuclear collapse of mitochondria observedupon both deletion and overexpression of Myo19 could be inducedby an imbalance of Miro-mediated microtubule-dependenttransport. Deletion of Myo19 might affect the equilibrium ofkinesin- and dynein-dependent mitochondrial movements, whereasits overexpression might compete differentially with these twomotors. Similarly to the deletion of Myo19, deletion of Miro1/2caused perinuclear accumulation of mitochondria. The search fornearest neighbours of the Myo19 tail region using BioID did notreveal any known binding partners of Miro1/2. However, additionalproteins were identified that are associated with the mitochondrialouter membrane, such asMAVS (mitochondrial anti-viral signallingprotein) and metaxin-3. The connection of these proteins to Myo19should be investigated further.

Intriguingly, downregulation of the Myo19 receptors Miro1/2, oroverexpression of either TRAK1 or TRAK2 caused concomitantreduction of Myo19 protein. The binding of Myo19 to Miro1/2 on

Fig. 4. Overexpression of Myo19 tail,TRAK1 or TRAK2 induces the loss ofendogenous Myo19. (A,B) HeLawild-type (wt) and stably transfectedmyc-BirA*-Myo19 tail (tail) cells wereincubated overnight with sodiumbutyrate to enhance expression of therecombinant construct. Cells were lysedand separated into cytosolic andmitochondrial fractions. (A) Immunoblotsfrom a representative experiment.Myo19 is strongly reduced in themitochondrial fraction of myc-BirA*-Myo19 tail-expressing cells and is notdetectable in the cytosol. GAPDHserves as a cytosolic and VDAC1 as amitochondrial marker, respectively.(B) Quantification of band intensities forMyo19, TRAK1 and Miro1/2 in themitochondrial fraction. Band intensitiesin the mitochondrial fractions werenormalized to wt (black bars) and thefold changes are shown for the tail-expressing cells. Data are representedas mean±s.e.m., n=3. (C,D) HEK293Tcells were transiently transfected withmCherry, mCherry-TRAK1, mCherry-TRAK2, mCherry-TRAK1-C (residues396-953) or mCherry-TRAK2-C(residues 324-913), respectively.(C) Cell homogenates were prepared24 h after transfection andimmunoblotted with the indicatedantibodies. For detection of mCherry-TRAK constructs two different blots wereassembled, as indicated by a dashedline. (D) Miro1/2 and Myo19 proteinlevels were quantified in relation toVDAC1. Levels of Myo19 aresignificantly reduced upon transientoverexpression of TRAK constructs.Data are represented as mean±s.e.m.,n=4-7.

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mitochondria stabilizes the protein and the loss of this interactionleads to Myo19 destabilization and degradation. Miro1/2 can bedownregulated by the two Parkinson’s disease genes PINK1and Parkin, which participate in mitochondrial quality control(Chan et al., 2011; Wang et al., 2011). Additionally, thedegradation of Miro2 is regulated by the PGAM5-KEAP1-Nrf2complex. Miro2, but not Miro1, is a substrate for KEAP1–cullin-3E3 ubiquitin ligase and the proteasome (O’Mealey et al.,2017). Following Miro1/2 degradation by either pathway,microtubule-based mitochondrial movement is halted (Wanget al., 2011; O’Mealey et al., 2017). Similarly, actin-basedmotility via Myo19 will be abrogated by loss of Miro1/2. Inagreement with our work, López-Doménech et al. (2018) reportedin an independent study that Miro1/2 stabilize Myo19 onmitochondria. A remarkable analogy represents vacuole inheritancein S. cerevisiae that relies on actin-based transport by the class Vmyosin Myo2. Unloading of the vacuole at its proper destinationfrom Myo2 is controlled by the degradation of its vacuole adapterVac17 (Yau et al., 2017).

Recently, it has been reported that a peptide encompassingresidues 860-890 in the tail region of Myo19 serves as an outermitochondrial membrane-binding motif (Shneyer et al., 2016;Hawthorne et al., 2016). This finding agrees well with ourobservation that an N-terminal tail fragment (aa 824-897) ofMyo19 is able to localize to mitochondria. However, this lipid-binding motif alone appears not to provide sufficient specificity andfunctionality under physiological conditions. To establish specificityand fully functional association with mitochondria, Myo19 has tointeract additionally with Miro. Although the overexpressed Myo19Miro-binding motif appeared to be localized mainly in the cytosol, itinduced perinuclear collapse of the mitochondria, indicating thatit successfully competed with other proteins for binding to Miro.An increase of the Miro1/2 concentration resulted in the distinctrecruitment of theMiro1/2 binding C-terminal tail fragment (aa 898-970) of Myo19 to mitochondria (data not shown). Miro1/2 were notonly necessary, but also sufficient to recruit the Myo19 tail. WhenMiro1/2 were redirected to early endosomes, theMyo19 tail was alsolocalized on early endosomes. Furthermore, this result demonstrates

Fig. 5. The Myo19 tail contains a Miro1/2-independent and a Miro1/2-dependent mitochondrial targeting site. (A) HeLa cells were transfected withEGFP-Myo19-tail (aa 824-970), EGFP-Myo19 N-tail (aa 824-897) or EGFP-Myo19 Myo19 C-tail (aa 898-970) constructs together with mTagBFP2-TOMM20-N-10 and mCherry-MiroΔTM-EE [2xFYVE early endosome targeting sequence (EE)] as indicated. Merged images are shown in colour on the right. (B,C) Statisticalanalysis of the correlation between the localization of Myo19 tail constructs (Tail, N-tail, C-tail) with Miro1ΔTM-EE (B) and TOMM20 (C), respectively.(D-F) Amino acid residues 1-408 of Miro-1 are sufficient to recruit the Myo19 tail and C-tail constructs. (D) HeLa cells were transfected with indicated constructs.Merged images are shown in colour on the right. (E,F) Statistical analysis of the correlation between the localizations of Myo19 tail constructs (Tail, N-tail, C-tail)with Miro1-aa1-408-EE (E) and TOMM20 (F), respectively. (G-I) The nucleotide state of the N-terminal GTPase domain of Miro regulates the mitochondrialrecruitment of the Myo19 tail and C-tail constructs. (G) HeLa cells were transfected with indicated constructs. Merged images are shown on the right. (H,I)Statistical analysis of the correlation between the localization of Myo19 tail constructs with Miro1(T18N)ΔTM-EE (H) and TOMM20 (I), respectively. In box-plots,square indicates the sample mean, the crosses the 1st and 99th maximum values, the dashes the minimum and maximum values, respectively. n≥30;****P≤0.0005, ***P≤0.001, *P≤0.05, n.s., not significant test. Scale bars: 20 µm.

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the predominance of the interaction with Miro1/2 over the lipidbinding by Myo19. In a recent report it was suggested that themitochondrial localization of Myo19 depends exclusively on thepresence of Miro1/2 (López-Doménech et al., 2018).We showed here that the N-terminal Rho-like GTPase domain of

Miro1/2 is critically involved in the recruitment of Myo19.Currently, there is no conclusive evidence available as to whether

this domain cycles between a GDP- and a GTP-bound state in vivo.Based on sequence comparisons, it appears likely that this domainin Miro1/2 has GTP bound constitutively, and is not cycling,since some residues important for hydrolysis are not conserved(Longenecker et al., 2003; Fransson et al., 2003; Li et al., 2002;Foster et al., 1996; Nobes et al., 1998). Marginal GTPase activityhas been reported for this domain in Gem1p, the yeast analogue,

Fig. 6. Myo19, Miro1/2 and Kif5B regulate thesubcellular distribution of mitochondria in HeLacells. (A) Western blots showing the depletion ofMiro1/2 and Kif5B (left panel) and Myo19 (right panel),respectively, 72 h after siRNA treatment comparedwith cells treated with non-targeting (nt) siRNA. Inthese experiments, the reduction of Miro1/2 was 95%,the reduction of Kif5B was 96%, and the reduction ofMyo19 was 97%. β-actin serves as loading control forequal amounts of total protein. Comparable resultswere obtained in all mitochondria distributionexperiments involving the depletion of Miro1/2, Kif5Bor Myo19. (B) HeLa cells were transfected with (fromtop to bottom) nt-siRNA as control or with siRNAsagainst Miro1/2, Kif5B or Myo19. OverexpressedGFP-Mito labels mitochondria, F-actin is stained withTexas Red-phalloidin, and nuclei are stained withDAPI. nt-siRNA transfected control cells exhibit normaldistribution of mitochondria, whereas depletion ofMiro1/2, Kif5B or Myo19 leads to clustering of themitochondria around the nucleus. Scale bar: 10 µm.(C) The observed perinuclear accumulation ofmitochondria due to depletion of Miro1/2, Kif5B orMyo19 was significant, as measured by theperinuclear accumulation factor. n=94-97 cells perconstruct from three independent experiments.***P≤0.001, **P≤0.01.

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with one GTP hydrolysed in 5-10 min (Koshiba et al., 2011).Phosphorylation of a serine residue was recently suggested toincrease GTPase activity (Lee et al., 2016). Mutation inMiro1/2 of athreonine residue at the end of the P-loop that is conserved innucleotide binding proteins to asparagine (T18N) abrogated therecruitment and stability of Myo19. Introduction of the analogousmutation (T19N) in RhoA produces a dominant-negative form. ThisRhoA mutant has an accelerated rate of GTP hydrolysis, but is

mostly in a nucleotide-free state exhibiting a high affinity forguanine nucleotide exchange factors (Strassheim et al., 2000;Miyazaki et al., 2002). Genetic experiments in Drosophila withthis Miro mutant suggested that it is essentially a recessive loss-of-function mutation (Babic et al., 2015). Residues mutated indominant active constructs of RhoA are not conserved in Miro1.Therefore, it is not certain whether the corresponding mutationsin Miro1 will induce similar alterations. Neuronal phenotypes

Fig. 7. Overexpression of GFP-labelled Myo19or tail fragments thereof causes perinuclearaccumulation of mitochondria in HeLa cells.(A) HeLa cells were co-transfected with (from topto bottom) GFP, GFP-Myo19, GFP-Myo19-tail,GFP-Myo19-C-tail or GFP-Myo19-N-tail andmCherry-Mito to label mitochondria. Nuclei arestained with DAPI. GFP-overexpressing controlcells exhibited normal distribution of mitochondria,whereas the mitochondria are clustered aroundthe nucleus in cells overexpressing Myo19 or tailfragments thereof. The effect is most pronouncedfor cells overexpressing GFP-My19-tail or GFP-Myo19-C-tail. Scale bar: 10 µm. (B) Theperinuclear accumulation factor was defined asthe percentage of mitochondria within a 4-µm-wide region around the nucleus, divided by thepercentage of cell area occupied by this region.Overexpression of any of the GFP-Myo19constructs increased the perinuclearaccumulation of mitochondria significantly.n=153-159 cells per construct from threeindependent experiments. ***P≤0.001, **P≤0.01.

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observed for one such point mutation in Drosophila (dMiroA20V)were not compatible with a constitutively active mutation (Babicet al., 2015). It remains to be determined whether cycling betweendifferent nucleotide states of the N-terminal GTPase domain ofMiro1/2 represents a physiological mechanism to regulate Myo19recruitment. The protein VopE from the bacterial pathogen Vibriocholerae has been proposed to act as a GTPase-activating protein(GAP) for the N-terminal GTPase domain of Miro1/2 and the

mammalian protein RAP1GDS1 as a guanine-nucleotide exchangefactor (GEF) (Suzuki et al., 2014; Ding et al., 2016).

Myo19 was shown to regulate the subcellular distribution ofmitochondria. Both downregulation and overexpression of Myo19induced a perinuclear accumulation of mitochondria. A similarperinuclear accumulation of mitochondria was observed in theabsence of Miro1 which negatively affected cytoplasmic energydistribution, cell migration and cell adhesion (Schuler et al., 2017).

Fig. 8. Perinuclear accumulation ofmitochondria following overexpression ofMyo19 or Myo19 tail fragments is independentof EGFP. (A) HeLa cells overexpressing (from topto bottom) Halo, Halo-Myo19, Myo19-Halo, Halo-Myo19-tail, or Myo19-tail-Halo are shown.Mitochondria are labelled by overexpressed GFP-Mito. Halo was labelled with a TMR-Halo ligandand nuclei were stained with DAPI. Halo-overexpressing control cells exhibit a normaldistribution of mitochondria, whereas themitochondria are clustered around the nucleus incells overexpressing Halo-Myo19-tail or Myo19-tail-Halo. Cells overexpressing full-length Halo-Myo19 or Myo19-Halo showed only weakclustering of the mitochondria around the nucleus.Scale bar: 10 µm. (B) Overexpression of Halo-Myo19-tail or Myo19-tail-Halo increased theperinuclear accumulation of mitochondriasignificantly, although the effect was morepronounced for Halo-Myo19-tail. A similarsignificance was observed for full-length Myo19-Halo, but not for Halo-Myo19. n=114-120 cells perconstruct from three independent experiments.***P≤0.001, *P≤0.05, n.s., not significant.

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During cell division, Myo19 regulates the allocation ofmitochondria to daughter cells. Loss of Myo19 causes asymmetricpartitioning of the mitochondria to one or both spindle poles inmitosis and stochastic failure of cytokinesis (Rohn et al., 2014).A similar phenotype in mitochondria segregation during mitosiswas recently reported for the loss of Miro1/2 (López-Doménechet al., 2018). In the presence ofMyo19, mitochondria do not localizeto spindle microtubules, but instead are localized in the periphery(Lee et al., 2007; Chung et al., 2016). Forced attachment of dyneinor kinesin motors to the mitochondrial surface during mitosisgenerates a phenotype comparable to that observed afterdownregulation of Myo19 (Chung et al., 2016; Rohn et al., 2014).These findings further hint at a role for Myo19 in the regulation ofmicrotubule-based transport of mitochondria. Therefore, it will beinteresting to elucidate further the interplay between Miro1/2 andMyo19 in the control of microtubule- and actin-based transport ofmitochondria.

MATERIALS AND METHODSReagents and antibodiesThe antibodies used were as follows: Myo19 (EPR12551-13/ab174286,Abcam, WB: 1:1000; HPA021415, Sigma-Aldrich, WB: 0.18 μg/ml);Miro-1/2 (NBP1-59021, Novus Biologicals, WB: 0.25 μg/ml); KIF5B(ab167429; Abcam, WB: 0.14 μg/ml); TRAK1 (PA5-44180, ThermoFisher, WB: 0.5 μg/ml); VDAC1 (20B12AF2/ab14734, Abcam, WB: 1 μg/ml); myc (ab9106, Abcam, WB: 0.04 μg/ml); GAPDH (TA802519;OriGene, WB: 0.5 μg/ml); β-actin (AC-15/A1978, Sigma-Aldrich, WB:1 μg/ml); mCherry 1C51 (ab125096 Abcam, WB: 0.5 μg/ml); Goat Anti-Rabbit IgG (H+L)-HRP (111-035-003, Jackson ImmunoResearch, WB:0.16 μg/ml); Goat Anti-Mouse IgG (H+L)-HRP (115-035-003, JacksonImmunoResearch, WB: 0.16 μg/ml); Streptavidin-HRP (016-030-084,Dianova, WB: 0.2 μg/ml);

Transfection reagents used in this study were as follows: LipofectamineLTX & PLUS Reagent (15338100, Thermo Fisher Scientific);Lipofectamine RNAiMAX Reagent (13778150, Thermo Fisher Scientific);PolyFect Transfection Reagent (301105, Qiagen); Nanofectin Kit(Q051-005, PAA Laboratories); polyethylenimine (PEI 25K, 23966-1,Polysciences); and ScreenFect A (299-73203, Wako Laboratory Chemicals).

The following siRNAs were used (5′-3′): siRNA Myo19 (GCAAAUG-ACUGGAGCCGCA, UAACAACAGCAGUCGCUUU, GGGAGGUCC-UGCUGUACAA, CGCCCGAGCUAAUGAGAGA (L-017137-01-0005,Dharmacon, GE Healthcare); siRNA non-targeting (UGGUUUACAUGU-CGACUAA, UGGUUUACAUGUUGUGUGA, UGGUUUACAUGUUU-UCUGA, UGGUUUACAUGUUUUCCUA (D-001810-10, Dharmacon,GE Healthcare); siRNA Miro-1 (#-09 UGUGGAGUGUUCAGCGAAA,#-10 GCAAUUAGCAGAGGCGUUA, #-11 CCAGAGAGGGAGACAC-GAA, #-12 GCUUAAUCGUAGCUGCAAA (J-010365-XX, Dharmacon,GE Healthcare); siRNA Miro-2 (#-09 GCGUGGAGUGUUCGGCCAA,#-10 CCUCAAGUUUGGAGCCGUU, #-11 GAGGUUGGGUUCCUGA-UUA, #-12 AGGAGAUCCACAAGGCAAA, J-008340-XX, Dharmacon,GE Healthcare); SMARTpool Kif5B siRNA (Dharmacon: ON-TARGET-plus KIF5B siRNA L-008867-00-0005).

Construction of plasmidsHuman Myo19 cDNA (AK304073/FLJ61052/TRACH 3006685) wasobtained from the National Institute of Technology and Evaluation(NITE) Japan. It was subcloned into pEGFP-C1 by PCR amplification ofa 5′- and a 3′-fragment, introducing a BglII and an XbaI site, respectively.The plasmid pEGFP-Myo19 tail codes for a fusion protein of EGFP withamino acids 824-970 of Myo19, pEGFP-Myo19 N-tail with amino acidresidues 824-897 of Myo19 and pEGFP-Myo19 C-tail with amino acidresidues 898-970 of Myo19, respectively. The myc-BirA R118G-MCS(BioID) plasmid was obtained from Addgene (plasmid #35700; Roux et al.,2012). The Myo19 tail cDNA coding for residues 824-970 was subclonedinto the XhoI and HindIII restriction sites. To construct pMyo19-Halo, theHalo-tag flanked by a BshTI restriction site and a stop codon followed by a

NotI restriction site was amplified by PCR using plasmid pFN21AA1042(Kazusa DNA Research Institute) as a template. The Halo-tag cDNA wasused to replace the cDNA for EGFP in pMyo19-EGFP-N1. pHalo-Myo19was constructed by replacing EGFP in the plasmid pEGFP-C1-Myo19 withamplified Halo-tag using the restriction sites BshTI and BglII. PlasmidpMyo19tail-Halo-N1 was constructed by amplifying Myo19tail frompEGFP-C1-Myo19tail by PCR, introducing BglII and EcoRI restrictionsites. This PCR product was then used to replace the Myo19 codingsequence in pMyo19-Halo-N1. Plasmid pHalo-C1-Myo19tail wasconstructed by amplifying Halo from plasmid pFN21AA1042 by PCR,introducing AgeI and BspEI restriction sites. Using these sites, this PCRproduct was then used to replace the EGFP coding sequence in pEGFP-C1-Myo19tail-avi-FLAG. To express GST-Myo19 C-tail (aa 898-970)-FLAG,the sequence coding for C-tail-FLAG was amplified by PCR and subclonedinto pGEX-4T-1 using EcoRI/SalI restriction sites. pRK5-Myc-Miro-1 andpRK5-myc-Miro-2 plasmids were gifts from Pontus Aspenström,Department of Microbiology, Tumor and Cell Biology, KarolinskaInstitutet, Stockholm, Sweden (Fransson et al., 2003). The plasmidpRK5–myc-Miro2 T18N was obtained from Addgene. The plasmidmCherry-Miro-1 was constructed by inserting Miro-1 into the XhoI/SacIIsites of pmCherry-C1. The point mutation to create plasmid mCherry-Miro-1 N18 was introduced by QuikChange mutagenesis. The GFP-2xFYVEHrs

plasmid was a gift from Marco Falasca, Centre for Cell Biology andCutaneous Reserch, London, UK (Maffucci et al., 2003). The plasmidmCherry-Miro-1-ΔTM-2xFYVE has the sequences coding for amino acidresidues 592-618 of Miro-1 replaced by the sequences coding for 2xFYVEdomains. The plasmid coding for the fusion protein pmCherry-Miro-1-aa1-408-2xFYVE was constructed by standard molecular biology techniques.The pET28a(+) plasmid encoding hMiro1-ABC(aa1-592)-6xHis was a giftfrom Sarah E. Rice, Department of Cell Biology and Molecular Biology,Chicago, USA (Klosowiak et al., 2016). Plasmid pFN21AA1042 coding forHalo-tagged human TRAK1 was obtained from Kazusa DNA ResearchInstitute. The cDNA sequence coding for TRAK1 was subcloned intopmCherry-C1 using XhoI and SalI restriction sites. The plasmid coding formCherry-TRAK1 amino acid residues 396-953 (TRAK1-C) wasconstructed using PCR. Plasmid pCMV-4a rat TRAK2-FLAG was agenerous gift from A. Stephenson (University College London, UK). TheTRAK2 cDNA encoding either full-length TRAK2 or residues 324-914(TRAK2-C) were subcloned into pmCherry-C1 vector using XhoI andEcoRI restriction sites. The plasmid encoding the mitochondrial targetingsequence from the subunit VIII of the human cytochrome c oxidase fused toRFP was purchased from BD Pharmingen and the sequence coding for RFPwas exchanged for EGFP and mCherry, respectively. Plasmid mTagBFP2-TOMM20-N-10 was from Addgene (plasmid #55328, deposited byMichael Davidson; Subach et al., 2011). All fragments amplified by PCRwere sequenced.

Cell culture and transfection of HeLa cellsHeLa and HEK 293T cells (ATCC) were cultivated in DMEMsupplemented with 10% (v/v) FCS, 100 U/ml penicillin and 100 μg/mlstreptomycin (complete DMEM) at 37°C, 95% humidity and 5% CO2. Fortransfection of HeLa cells, 20,000 cells were seeded onto glass coverslips in24-well plates. Cells were transfected 1 day after seeding as described in themanual of the manufacturer. Briefly, 0.5 µg of the plasmid DNAwas mixedwith 1 μl Lipofectamine LTX and 0.5 µl PLUS Reagent in 50 μl DMEM,vortexed, incubated at room temperature (RT) for 5 min and added drop-wise to the cells. For cotransfection of two plasmids, the amount of plasmidDNA was reduced to 0.25 µg for each plasmid. After 4 h of incubation theDNA-lipid complexes were removed, cells were washed with PBS and freshcomplete DMEM was added. Cells were fixed 24 h after transfection.

For live cell imaging, ∼40,000 HeLa cells were seeded onto a 35 mmµ-dish (ibidi) in 1 ml complete DMEM 1 day before transfection. Fortransfection, 2 µg of the plasmid DNA in 50 μl diluent (150 mMNaCl) wasmixed with 6.4 μl Nanofectin in 50 μl diluent, and vortexed. The mixturewas incubated at RT for 30 min and added to the µ-dish drop wise. Thetransfection complexes were removed after 4 h of incubation, and freshcomplete DMEM was added to the µ-dish. After 24 h of incubation, theexpression of recombinant proteins was analysed.

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To generate the stable HeLa myc-BirA* Myo19 tail, Halo-Myo19 andMyo19-Halo cell lines, 100,000 cells per well were seeded in a 6-well plate.The following day, 2.5 µg of the plasmid DNA with 5 μl LipofectamineLTX and 2.5 µl PLUS Reagent in 250 μl DMEM was used for transfection.After 48 h, complete DMEM was supplemented with 600 µg/ml G418and 60%, 30% and 10% of cells, respectively, were replated in 150-mm-diameter cell culture dishes. Cells still viable after 2 weeks were consideredstably transfected. Single colonies of cells were isolated with a metal ring,trypsinized and transferred to 6-well plates. Cells were processed for westernblot analysis and further expansion.

For knockdown studies of Miro-1 and Miro-2, 100,000 HeLa cells perwell were seeded in 6-well plates. The following day, cells in one well weretransfected with 25 pmol of each siRNA or each pool of 4 siRNAs thatwas mixed with 7.5 μl Lipofectamine RNAiMAX in 183 μl DMEM. Cellswere harvested for western blot analysis 48 h and 72 h after transfection.Transient transfections of HEK cells were performed in 6-well plates byseeding 150,000 cells per well. The following day 4 μg of plasmid DNA in100 μl DMEM were mixed with 12 μg PEI in 100 μl DMEM, vortexed,incubated for 30 min at RT and then added drop-wise to the cells. Cellswere harvested 24 h after transfection and processed for immunoblotanalysis.

BioIDBioID was carried out essentially as described in Roux et al. (2013) withsome modifications. Cells were incubated for 24 h in complete DMEMsupplemented with 50 µM biotin before lysis and affinity purification ofbiotinylated proteins. To enhance expression of the construct, 10 mMsodium butyrate was added to the medium 15 h before harvesting ofthe cells.

Approximately 2×108 stably transfected myc-BirA* Myo19 tail HeLacells (10 confluent 150-mm-diameter cell culture dishes) were washed threetimes with cold PBS and collected by scraping. They were resuspended in20 ml lysis buffer [50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.2% (w/v)SDS, 0.1 mg/ml Pefabloc, 0.01 mg/ml Leupeptin and 0.02 U/ml Aprotinin]before 2 ml of 20% (v/v) Triton X-100 were added. The subsequent stepswere performed at 4°C. Lysates were sonicated with two 30 pulses using anamplitude of 100% and a proportional sonication period of 0.6 (UP 100Hsonicator, Hielscher Ultrasound Technology). Prior to the second sonicationcycle 20 ml of 50 mM Tris-HCl, pH 7.4, were added. Lysates were clarifiedby centrifugation at 16,600 g for 10 min. Supernatants were incubated with8 mg of equilibrated streptavidin magnetic beads (Pierce 88817, ThermoFisher Scientific) overnight. Beads were collected and washed with washbuffer 1 [2% (w/v) SDS in ddH2O] for 8 min at RT. This step was repeatedwith wash buffer 2 [0.1% (w/v) deoxycholic acid, 1% (w/v) Triton X-100,1 mM EDTA, 500 mM NaCl and 50 mM Hepes, pH 7.5], wash buffer 3[0.5% (w/v) deoxycholic acid, 0.5% (w/v) NP-40, 1 mM EDTA, 250 mMLiCl and 10 mM Tris-HCl, pH 7.4] and wash buffer 4 (50 mM Tris-HCl,pH 7.4), respectively.

Biotinylated proteins were eluted from the beads at 98°C for 8 min with200 µl buffer containing 40 mM Tris-HCl, pH 6.8, 1 mM EDTA, 8% (w/v)sucrose, 3% (w/v) SDS, 2% (w/v) 2-mercaptoethanol, 0.004% (w/v)bromophenol blue and 820 µM biotin. Aliquots of the eluates were analysedby immunoblotting (20 µl) and by Coomassie Blue staining followingSDS-PAGE (40 µl). Isolated bands were subjected to mass spectrometry(University Hospital Cologne, Centre of Molecular Medicine (ZMM),Central Bioanalytics (ZBA)).

Expression and purification of recombinant proteinsMiro1 (aa 1-592, C-terminal 6xHis-tag) (Miro1-ABC) was expressed inE. coli Rosetta 2 (DE3) cells and purified by metal ion affinitychromatography with TALON beads (Clontech Laboratories, Inc.). Cellswere cultured in TPM medium (20 g/l tryptone, 15 g/l yeast extract, 8 g/lNaCl, 2 g/l Na2HPO4, 1 g/l KH2PO4) containing 50 µg/ml kanamycin and34 µg/ml chloramphenicol. Protein expression was induced overnight at18°C with 125 µM IPTG when cell density had reached an OD600 of 0.9.Cells were harvested by centrifugation (4400 g, 15 min, 4°C), washed withcold PBS (137 mM NaCl, 2.7 mM KCl, 10.2 mM Na2HPO4, 1.8 mM

KH2PO4, pH 7.0) and stored at−80°C. For protein purification, cell pelletswere resuspended in lysis buffer (25 mM Hepes, pH 7.4, 300 mM NaCl,0.5 mM TCEP, 8 mM imidazole, 2 mM MgCl2, 5% sucrose, 0.02%Tween-20, 0.1 mg/ml Pefabloc®, 0.01 mg/ml Leupetin, 0.02 U/mlAprotinin, 2 mM ATP) and cells were lysed by sonication for 4×2 minwith 2 min intervals using an amplitude of 100% and a proportionalsonication period of 0.6 (UP 100H sonicator, Hielscher UltrasoundTechnology). The lysate was clarified by ultracentrifugation (139,000 g,45 min, 4°C) and batch adsorbed to pre-equilibrated TALON beads for 1 hat 4°C. The beads were collected and washed three times with washingbuffer (25 mM Hepes, pH 7.4, 300 mM NaCl, 0.5 mM TCEP, 12 mMimidazole, 2 mM MgCl2, 5% sucrose, 0.02% Tween-20) before they weretransferred to a gravity-flow column. Bound protein was eluted withelution buffer (25 mM Hepes, pH 7.4, 300 mM NaCl, 0.5 mM TCEP,300 mM imidazole, 2 mMMgCl2, 5% sucrose, 0.02% Tween-20), pooledand stored at −20°C.

Myo19 C-tail (aa 898-970) fused to a N-terminal GST-tag and aC-terminal FLAG-tag was expressed in E. coli Rosetta 2 (DE3) cells andpurified by glutathione affinity chromatography followed by FLAG-antibody affinity chromatography. The procedure for cell growth,induction of protein expression and harvest of cells was as describedabove for Miro1-ABC protein purification. Cell pellets were storedovernight at −80°C. They were resuspended in lysis buffer (20 mMHepes, pH 7.4, 100 mM NaCl, 50 mM KCl, 2 mM MgCl2, 10% glycerol,1 mM 2-mercaptoethanol, 0.1 mg/ml Pefabloc®, 0.01 mg/ml Leupetin,0.02 U/ml Aprotinin, 2 mM ATP) and lysed by sonication. Followingultracentrifugation (139,000 g, 45 min, 4°C), the supernatant was incubatedwith pre-equilibrated glutathione Sepharose beads. The beads werecollected and washed three times with washing buffer (20 mM Hepes, pH7.4, 100 mM NaCl, 50 mM KCl, 2 mMMgCl2, 1 mM 2-mercaptoethanol).The protein bound to the resin was eluted by cleavage of the GST-moiety bythe addition of 40 U of thrombin overnight in washing buffer. Beads werecentrifuged at 700 g for 5 min at 4°C and washed twice. The supernatantswere combined and passed several times over FLAG-antibody agarose(ANTI-FLAG® M2 Affinity Gel, Sigma-Aldrich). The column resinwas washed with washing buffer, followed by two alternating high salt(20 mM Hepes, pH 7.4, 450 mM NaCl, 50 mM KCl, 2 mM MgCl2, 1 mM2-mercaptoethanol) and low-salt (20 mM Hepes, pH 7.4, 50 mM KCl,2 mM MgCl2, 1 mM 2-mercaptoethanol) washes, followed by washingbuffer. Finally, Myo19 C-tail was eluted with 0.25 mg/ml FLAG®-peptide(Sigma-Aldrich) in washing buffer.

Pull-down assayPurified hMiro1-ABC was loaded with GTP by adding 2 mM GTP and6 mM EDTA for 10 min at RT followed by the addition of 15 mM MgCl2.Next, the protein was diluted with assay buffer (25 mM Hepes, pH 7.4,300 mM NaCl, 0.5 mM TCEP, 2 mM MgCl2, 5% sucrose, 0.02% Tween-20) to a final concentration of 10 mM imidazole and adsorbed to pre-equilibrated TALON beads at 4°C. Beads were washed three times withwashing buffer (25 mM Hepes, pH 7.4, 300 mM NaCl, 0.5 mM TCEP,12 mM imidazole, 2 mM MgCl2, 5% sucrose, 0.02% Tween-20) andtransferred to a gravity-flow column. Myo 19 C-tail was passed repeatedlyover the column before it was washed three times with washing buffer.Adsorbed Miro1-ABC was eluted with an excess of imidazole in the buffer(25 mMHepes, pH 7.4, 300 mMNaCl, 0.5 mM TCEP, 400 mM imidazole,2 mM MgCl2, 5% sucrose, 0.02% Tween-20). Eluted proteins wereanalysed by SDS-PAGE and immunoblotting.

Preparation of cell homogenatesCells grown in 6-well plates were washed three times with cold PBS andgently scraped off. They were centrifuged at 600 g at 4°C for 5 min andresuspended in 100 µl lysis buffer [50 mM Tris-HCl pH 7.4, 2 mMMgCl2,100 mMNaCl, 1% (v/v) NP-40, 10% (v/v) glycerol, 1 mMDTT, 0.1 mg/mlPefabloc, 0.01 mg/ml Leupeptin and 0.02 U/ml Aprotinin]. Proteinconcentrations were determined by Bradford assay using BSA as astandard. Equal amounts of protein were separated by SDS-PAGE andfurther processed for immunoblotting.

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Pulse-chase experimentsFor pulse-chase experiments, stable Halo-Myo19 or Myo19-Halo HeLa cellswere transfected with siRNA for Miro1/2. 24 h later, the cells were pulselabelled with 2 µM HaloTag® TMR ligand (Promega) for 30 min at 37°C,97% humidity and 5% CO2. Free ligand was removed by several washingsteps and a 30 min incubation with complete DMEM supplemented with600 µg/ml G418. Next, the cells were incubated with 5 µM HaloTag® Biotinligand, to prevent further labelling of the Halo-tag with any remainingfluorescent ligand. Cells were harvested at 0, 4, 8 and 12 h time points.Proteins were separated by SDS-PAGE, electrophoretically transferred to aPVDF membrane and analysed for TMR fluorescence followed byimmunoblotting. Fluorescent bands were quantified using ImageJ.

Mitochondria purificationPrior to mitochondria purification, HeLa wt and HeLa myc-BirA* Myo19tail cells were treated overnight with 10 mM sodium butyrate. Mitochondriawere purified with Qproteome Mitochondria Isolation Kit (QIAGEN)according to the manufacturer’s protocol for high-purity mitochondria.

Immunofluorescence stainingTo label mitochondria, cells were incubated with 75 nM MitoTracker RedCMXRos (M7512, Thermo Fisher Scientific) in complete DMEM at 37°C,95% humidity and 5% CO2 for 10 min. Immediately afterwards, cellswere washed three times with warm PBS and fixed with 4% (w/v)paraformaldehyde (PFA) in PBS at 37°C for 25 min. Free aldehydes werequenched with 0.1 M glycine in PBS at RT for 10 min. Subsequently, cellswere washed three times with PBS and mounted with Mowiol [3.4 mMMowiol 4-88, 105 mM Tris-HCl, pH 8.5, 18.4% (v/v) Glycerin and223 mM DABCO]. To stain cells for endogenous Myo19, they werepermeabilized at RT for 15 min with 0.1% (v/v) Triton X-100 in PBS,followed by washing with PBS for 2×5 min. Unspecific binding sites wereblocked by incubation with blocking buffer [5% (v/v) normal goat serum inPBS] at RT for 10 min. The primary antibody (HPA021415, Sigma-Aldrich) was diluted 1:100 (1.8 μg/ml) in blocking buffer. Coverslips wereplaced on a drop of antibody solution in a humid chamber and incubatedovernight at 4°C. Thereafter, cells werewashed 3× for 5 min with PBS at RTand incubated in the dark with secondary antibody (111-545-003, JacksonImmunoResearch) diluted 1:500 (3 μg/ml) in blocking solution for nolonger than 1 h at RT. Subsequently, cells were washed three times withPBS and mounted with Mowiol [3.4 mM Mowiol 4-88, 105 mM Tris-HCl,pH 8.5, 18.4% (v/v) Glycerin, 223 mM DABCO].

Transferrin endocytosis assayHeLa cells grown on coverslips were serum-starved for 30 min in MEMmedium at 37°C, 5% CO2 and 95% humidity. AF 488-conjugated humantransferrin (30 µg/ml) (ThermoFisher) in MEM medium was added for 1 hat 37°C. Cells were washed with ice cold 1× PBS and subsequently fixedwith 4% PFA for 20 min at RT.

Retargeting and quantificationHeLa cells were fixed 24 h after transfection and analysed by spinning diskmicroscopy (UltraVIEW VoX, PerkinElmer; Nikon Eclipse Ti). Imageswere acquired with a 60×/1.49 NA oil objective and an EMCCD camerausing Volocity software. Z-stacks covering the entire depth of cells withintervals of 0.5 μm were acquired. To quantify retargeting of tail constructsto early endosomes, maximum intensity projections were obtained usingImageJ. To separate mitochondria from background a ‘top hat’ filter wasapplied. Pearson’s correlation coefficient was used to express the intensitycorrelation of co-localizing objects. Analysis was restricted to custom ROIof each cell, with a thresholding factor of 2. The result is +1 for perfectcorrelation, 0 for no correlation and −1 for perfect anti-correlation.

Mitochondria distributionTo assess mitochondria distribution, 20,000 HeLa cells per well were platedon glass coverslips in 24-well plates. The next day, cells were transfected withappropriate plasmids using the transfection reagent Lipofectamine™LTX andPLUS™ reagent (Invitrogen) according to the manufacturer’s instructions.

24 h after transfection, the cells were processed further: Cells that had beentransfected with Halo constructs were labelled with 5 µM HaloTag® TMRligand (Promega) in full medium for 30 min at 37°C, followed by severalwashing steps with PBS and full medium before fixation. Cells were washedwith PBS and fixed with 4% PFA, stained for DNA with DAPI and, ifappropriate, for F-actin with fluorophore-labelled phalloidin, mounted onmicroscope slides and left to harden at 4°C overnight. For experimentsinvolving protein depletion, 100,000 HeLa cells per well were seeded in 6-well plates. 24 h after seeding, the cells were transfected with appropriatesiRNAs using the transfection reagent Lipofectamine™ RNAiMAXaccording to the manufacturer’s instructions. The next day, the cells werereplated into 24 well plates and transfected, fixed and stained as describedabove. Imaging was performed with a 63× N.A. 1.4 Plan-Apochromat oilimmersion objective on a Zeiss LSM 510 confocal laser scanning microscopeequippedwithArgon andHelium-Neon lasers and three 12-bit R/FI detectors.Z-stacks of mitochondria and, if appropriate, other labelled structures wererecorded at a plane interval of 0.5 µm; upper and lower boundaries of thestacks were set manually for each field of view. Bright-field images wererecorded with 12-bit R/FI detectors along with the z-stacks. DAPIfluorescence was excited with a mercury vapour short-arc HBO 50 lampand detected with an 8-bit AxioCam MRm camera.

AnalysisImage processing and analysis were performed in ImageJ (NIH, USA)with customized macros. Briefly, z-stacks of fluorescence channels wereconverted to sum projections, and sum projections of the mitochondria weretop-hat filtered and background corrected. Size and orientation of the DAPIimages were adjusted to the confocal images. For each cell, the outline wasdetermined manually based on the bright-field image. Shape descriptorsroutinely available in ImageJ were measured for the cell outline. Thenucleus outline was determined via automated thresholding of the DAPIimage and was dilated to determine the boundary of the perinuclear region,which was defined as a 4 µmwide ring around the nucleus.When the dilatednucleus outline exceeded the cell outline, it was cut accordingly. For each ofthe cell’s regions (in/above/below nucleus, perinuclear, peripheric), theproportion of mitochondria signal wasmeasured as well as the fraction of thetotal cell area occupied by this region. The perinuclear mitochondriaaccumulation factor was defined as the proportion of mitochondrial signalwithin the perinuclear region, divided by the fraction of the total cell areaoccupied by the perinuclear region.

Statistical evaluationData are displayed as box plots with the box ranging from 25th to 75thpercentiles, whiskers ranging from 10th to 90th percentiles, the medianmarked as a line, and the mean marked as a square. Data were analysed byKruskal-Wallis ANOVA to locate statistically significant differences in setsof more than two samples followed by a Mann-Whitney U-test for pairwisecomparisons; the significance levels were set to *P≤0.05, **P≤0.01 and***P≤0.001.

AcknowledgementsWe are grateful to P. Aspenstrom, M. Falasca, A. Stephenson and S. E. Rice for thegift of plasmids, M. Horsthemke for help with artwork and to P. Hanley for valuablecomments on the manuscript. A preliminary account of this work was presented atthe 2016 ASCB Annual Meeting.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: S.J.O., K.M., X.-P.H., V.S.; Methodology: S.J.O., K.M., X.-P.H.,V.S., A.F., U.H.; Software: V.S.; Validation: K.M., X.-P.H., V.S.; Investigation: S.J.O.,K.M., X.-P.H., V.S., A.F., U.H., P.N.; Resources: A.F., U.H., P.N.; Data curation:S.J.O.; Writing - review & editing: S.J.O., K.M., X.-P.H., V.S.; Visualization:S.J.O., K.M. Supervision: M.B.

FundingThis work was supported by the Deutsche Forschungsgemeinschaft (BA 1354/10-1).K.M was supported by the Cells in Motion Excellence Cluster (CiM)/International

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Max-Planck Research School (IMPRS) graduate school and X.-P.H. by the NRWResearch School ‘Cell Dynamics and Disease, CEDAD’. S.J.O. acknowledgesthe receipt of a CiM bridging position.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.219469.supplemental

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