RESEARCH ARTICLE

Myo19 is an outer mitochondrial membrane motor and effector ofstarvation-induced filopodiaBoris I. Shneyer, Marko Usaj and Arnon Henn*

ABSTRACTMitochondria respond to environmental cues and stress conditions.Additionally, the disruption of the mitochondrial network dynamicsand its distribution is implicated in a variety of neurodegenerativediseases. Here, we reveal a new function for Myo19 in mitochondrialdynamics and localization during the cellular response to glucosestarvation. Ectopically expressed Myo19 localized with mitochondriato the tips of starvation-induced filopodia. Corollary to this, RNAinterference (RNAi)-mediated knockdown of Myo19 diminishedfilopodia formation without evident effects on the mitochondrialnetwork. We analyzed the Myo19–mitochondria interaction, anddemonstrated that Myo19 is uniquely anchored to the outermitochondrial membrane (OMM) through a 30–45-residue motif,indicating that Myo19 is a stably attached OMMmolecular motor. Ourwork reveals a new function for Myo19 in mitochondrial positioningunder stress.

KEY WORDS: Filopodia, Mitochondria, Myosin 19

INTRODUCTIONMitochondria are found in almost all eukaryotic cells and play a rolein processes such as ATP production, Ca2+ homeostasis, lipidsynthesis and apoptosis signaling (Nunnari and Suomalainen,2012). Mitochondria are organized as a network that undergoescontinuous events of fission and fusion, processes that are crucialfor their cellular function (Chen and Chan, 2009). The balancebetween the fission–fusion cycles is essential, as its disruption isimplicated in human neurodegenerative diseases (Lin and Beal,2006; Chen and Chan, 2009; Federico et al., 2012). Themitochondrial network responds to changes in physiologicalconditions, such as starvation and oxidative stress, as reflected inmorphological rearrangement that results in hyperfusion andfragmentation, respectively (Fan et al., 2010; Rambold et al.,2011). Additionally, the mitochondrial network responds to variouscues by changing its intracellular positioning, supplying local ATPdemand and providing a localized signaling platform (Tait andGreen, 2012; Desai et al., 2013; Sheng, 2014).Mitochondrial motility is primarily based on microtubules

(MTs), utilizing plus-end-directed kinesin motors and the minus-end-directed dynein (Pilling et al., 2006). However, the actincytoskeleton has also been shown to be involved in mitochondrialmotility, localization and cellular distribution (Bradley and Satir,1979; Chada and Hollenbeck, 2004). Myo19, an actin-basedmolecular motor, was discovered as a new mitochondria-localized

myosin in vertebrates. Myo19 localizes to mitochondria through itsC-terminal tail domain and nearly doubles the mitochondriamotility when ectopically expressed (Quintero et al., 2009). Thefinding of a myosin that localizes to the mitochondria suggests that itacts as the link between mitochondria and the actin cytoskeleton.Knockdown of Myo19 by RNA interference results in apparentdefects of mitochondrial segregation during division and incorrectmovement of the mitochondria to the spindle poles during anaphase(Rohn et al., 2014).

The interaction between the actin cytoskeleton, myosin motorsand their function in mitochondria dynamics, morphology andcellular localization is now beginning to emerge (Korobova et al.,2014). A new function for actin-based motors could emerge asregulators of cellular adaptations to stress, linking actin cytoskeletonremodeling and mitochondria (Jayashankar and Rafelski, 2014).These recent studies demonstrate an essential role for both actincytoskeleton and myosins for mitochondria function in homeostasisand pathological conditions.

To this end, we examined Myo19 involvement in themitochondrial network response to stress conditions induced inhuman U2OS cells. We discovered that Myo19 localizes withmitochondria to tips of starvation-induced filopodia in a process thatdepends on its ATPase activity and the actin cytoskeleton. We showthat RNA interference (RNAi)-mediated knockdown of Myo19attenuates the formation of starvation-induced filopodia. Deletion of45 amino acids in Myo19 tail domain (Myo19Δ851-895–eGFP)impairs its localization to the mitochondria. We further dissectedMyo19 binding to the mitochondria, revealing that it is a monotopicmembrane protein anchored to the OMM with both the N- and C-terminus facing the cytoplasm. To our knowledge, this is the firstvertebrate myosin that is directly bound to a membrane.

RESULTSGlucose starvation induced localization of mitochondria andMyo19 to foci at cell periphery protrusionsMitochondria function as an intracellular biosensor that responds toenvironmental changes, stress cues and physiological stimuli(Jayashankar and Rafelski, 2014). Hence, it is very intriguing totest the Myo19 interaction with the mitochondrial networkdynamics and morphology in cells under stresses, such asstarvation, that induce mitochondrial responses. We thereforeectopically expressed Myo19 fused to eGFP (Myo19–eGFP) inU2OS cells, and followed Myo19 and mitochondria localization inresponse to glucose starvation. Under complete medium conditions,Myo19 localized to mitochondria and there was also some diffusecytosolic appearance, which might be due to the ectopic expression,similar to in previous work (Fig. 1A). Notably, expression ofMyo19–eGFP caused mitochondria to clump together into globularstructures in a motor-dependent manner (Fig. S1A, white arrow;Fig. S1B). In contrast to complete medium conditions, glucosestarvation of U2OS cells resulted in the localization of Myo19Received 9 June 2015; Accepted 5 December 2015

Department of Biology, Technion Israel Institute of Technology, Haifa 3200003,Israel.

*Author for correspondence (arnon.henn@technion.ac.il)

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© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 543-556 doi:10.1242/jcs.175349

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Fig. 1. Glucose starvation induces mitochondria localization to foci in the cell periphery, which depends on an active Myo19. (A) Live imaging of U2OScells ectopically expressing the indicated Myo19 constructs grown in complete medium (CM) or under glucose starvation. White line, outline of a filopodiaemphasizing that both Myo19-eGFP and mitochondria are colocalized at filopodia tip. White arrow, example of Myo19 foci in the cellular protrusions. TheMag. image shows a magnification of the white-boxed region, showing that Myo19 and mitochondria are present in the foci. (B) Quantification of the focilocalization of the indicated Myo19 constructs. Myo19-expressing cells were scored according to the number of foci present under complete medium (containing25 mM glucose), starvation (no glucose), or starvation medium supplemented with 5.5 mM glucose. Values are presented as the percentage of cells having theindicated foci number out of the total number of analyzed cells (mean±s.d., n≥150 cells from three independent experiments). (C) Localization of ectopicallyexpressed Myo19 under starvation medium supplemented with 5.5 mM glucose. Note the lack of both peripheral protrusions and foci localization of Myo19(compare to row 2 in A). In A and C, blue, nuclei; DIC, differential interference contrast microscopy; green, eGFP-fused Myo19 constructs; red, mitochondria.Scale bars: 20 µm (10 µm in magnification).

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together with mitochondria to foci in protrusions extending from thecells (Fig. 1A). We quantified that >80% ofMyo19–eGFP foci werepositive for mitochondria by calculating the ratio between thenumber of MitoTracker-Red-stained Myo19–eGFP foci and thenumber of Myo19–eGFP foci (Fig. 1A, n=160 from 14 cells).Notably, these protrusions formed randomly around the cellperiphery, showing no preference of directionality, which is mostlikely due to missing cues such as mechanical forces or chemotacticmolecules (Petrie et al., 2009). The localization of Myo19 to thestarvation-induced foci depended on an active full-length Myo19(Fig. 1A). Neither eGFP alone, Myo19 tail (Myo19824–970–eGFP)nor ATPase-dead full-length Myo19 (Myo19G135R–eGFP) (Adikeset al., 2013) were able to localize to the protrusions, linking Myo19enzymatic function to protrusion localization. To test whether thelocalization required a mitochondria-binding motif, we repeated theexperiment with a cytosolic mutant of Myo19 (Myo19Δ851–895–eGFP), which was able to localize to these protrusions; however, nomitochondria colocalized with this deletion mutant, demonstratingthe requirement for mitochondria-binding motif for colocalizationwith the mitochondria (Fig. 1A). Verification of expression of theMyo19 constructs was performed by western blotting (Fig. S1C).Supplementation of the starvation medium with glucose completelyprevented both protrusions and foci formation, indicating that it is aresponse to glucose starvation rather than other nutrients (Venteret al., 2014) (Fig. 1C).We further examined the growth dynamics of these starvation-

induced protrusions and the emergence of Myo19 foci withinthem by performing live imaging of starved cells ectopicallyexpressing Myo19–eGFP (Fig. 2). Most of the foci formed withinthe cell boundary at time zero of imaging (+20 min of starvation),which is marked by yellow line (70%, n=100 from 14 cells),whereas fewer extended beyond the cell boundary (30%, n=100from 14 cells). However, we cannot conclude from theseexperiments the order of events between protrusion formationand Myo19 foci dynamics. The relationship between theprotrusions and Myo19 foci can also be seen in time-lapse DICimages, which clearly show the linkage between them (Fig. S2A).Similar protrusions were observed in wild-type (WT) cells understarvation (Fig. S2B; see also Fig. 5F).To this end, we demonstrate quantitatively the link between

Myo19, its mitochondria-binding motif and glucose starvation tolocalization to starvation-induced foci formation in the cell peripheryprotrusions. Moreover, these observations raised the notion thatMyo19might influence these protrusions and prompted us to test theinvolvement of the actin cytoskeleton in these structures.

The actin cytoskeleton is essential for starvation-inducedMyo19 foci formationTo support our results that an active motor is required for Myo19foci formation, we tested whether disruption of the actincytoskeleton would prevent their formation. Treating Myo19–eGFP-expressing cells with 0.2 µM latrunculin B (LatB) 30 minprior to starvation prevented the foci formation (∼70% of the cellsshowed less than two foci), further supporting that Myo19 fociformation is through the actin cytoskeleton (Fig. 3A). Alternatively,we show that nocodazole treatment of Myo19–eGFP-expressingcells induced Myo19 foci formation under complete mediumconditions (>80% of the cells). This is similar towhat is seen in cellsectopically expressing mDia1 and treated with nocodazole, whichshow a strong shift towards actin-based mitochondrial motility(Fig. 3B) (Minin et al., 2006). However, the molecular basis for thisis still unknown.

Starvation-induced protrusions possess filopodia markersWe had strong evidence that the foci are related to the actincytoskeleton. Therefore, we tested the starvation-inducedlocalization of Halo-tagged Myo19 (Myo19–Halo) with actin–eGFP in live cells or Alexa-Fluor-488–phalloidin-stained actin infixed cells, revealing that Myo19 foci localized at the tips of actinprotrusions (Fig. 4A, inset and line profile; Fig. S3A). Tocharacterize these actin protrusions, we tested Myo19 localizationwith focal adhesion and filopodia markers, as these structures mightprovide further insight on the nature of these protrusions. Vinculinand paxillin were used as markers to test whether Myo19 foci arerelated to focal adhesions. Ectopically expressed Emerald–vinculinlocalized to the base of the protrusions; however, Myo19 waslocalized further towards the tip (Fig. S3B, insets and line profile).Notably, expressing paxillin–eGFP inhibited protrusion formation,in accordance with published literature; however, it was notlocalized with Myo19 in the few protrusions that formed(Fig. S3C, insets and line profile).

The protrusions resembled filopodia in their structure, thereforewe tested Myo19 colocalization with the filopodia markers fascinand Drf3 (also known as mDia2), revealing that these actinprotrusions are indeed growing filopodia and that Myo19 is presentat their tip (Fig. 4B, inset, line profile). Glucose-starvation-mediatedinduction of filopodia formation is not limited to U2OS cells, as wealso show that this also occurs in the HeLa cell line (Fig. S3D),suggesting that this is a universal phenotype. This finding strongly

Fig. 2. Live imaging of starvation-induced foci localization. Micrographsfrom time-lapse live imaging of starved cells ectopically expressing Myo19–eGFP showing formation of Myo19 foci. Note that some of the Myo19 foci arelocalized within the cell boundaries (marked by a yellow line) at the beginning ofimaging (+20 min of starvation), whereas some foci extend beyond the cellboundary. Mag. image shows a magnification of the white-boxed region.

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encouraged us to test the contribution of Myo19 to filopodiaformation.

Myo19 is a de novo effector of starvation-induced filopodiaTo examine the contribution of Myo19 to filopodia formation, weutilized RNA interference to knockdown Myo19 from U2OS cellsand follow starvation-induced filopodia formation. We ectopicallyexpressed fascin–eGFP in these cells, allowing us to followfilopodia formation (Fig. 5A; Movie 1). Interestingly, filopodiaformed both at the dorsal side of the cell and at the cell periphery.However, Myo19 localized only to the peripheral filopodia, butnot to the dorsal filopodia, therefore only these were measured(Fig. 6A). To correctly measure the filopodia length using wide-field fluorescent microscopy, we verified that most of theperipheral filopodia were parallel to the plane by measuringtheir angle at the z-axis to be <2° (Fig. 6B). Evidentially,knockdown of Myo19 (∼80%, Fig. 5B) resulted in significantlyfewer (from 42±7.9 to 24±10.8 filopodia per cell, mean±s.e.m.)and shorter filopodia (from 2.9±1.22 µm to 1.9±0.74 µm inlength) (Fig. 5C,D). In addition, we quantified the filopodia lengthdistribution and observed a clear shift towards shorter filopodia inMyo19-knockdown versus mock-treated cells (Fig. 5E). The smallinterfering RNAs (siRNAs) utilized to knockdown Myo19 weretargeted against the 3′ UTR, allowing us to confirm that theeffects are specific to Myo19 by performing a rescue experiment,

where we knocked down Myo19 and ectopically expressed fascin–eGFP and Myo19–Halo. The rescue reversed the knockdown ofMyo19 as measured by restoration of starvation-induced filopodianumber to 39±6.5 filopodia per cell and length to 2.8±0.93 µm,similar to in mock-treated cells (Fig. 5D). Moreover, the rescuerestored the filopodia length distribution to a similar distributionto that of mock-treated cells (Fig. 5E).

Furthermore, we observed two differences between the mock-and Myo19-siRNA-treated cells. Mitochondria were present in aminority of the filopodia of mock-treated cells, which werecompletely absent from filopodia of Myo19 knockdown cells(Fig. 5F, arrow). Additionally, Myo19 knockdown cells had visiblepatches of fascin–eGFP at the cell periphery (Fig. 5G), which mightrepresent a possible failed filopodia formation site. Comparing therate of filopodia growth between mock-treated and Myo19-knockdown cells indicates that Myo19-knockdown cells feature apronounced longer lag before reaching a similar steady-state rate offilopodia growth (0.072±0.032 µm/min and 0.079±0.032 µm/min,for Myo19-knockdown and mock-treated cells, respectively),suggesting that Myo19 role is important for the dynamicdevelopment of filopodia (Fig. 6C).

We tested the effect of Myo19 knockdown on the mitochondrianetwork morphology; however, we did not notice any majorchanges compared to mock-treated cells (Fig. 5H). Our collectiveobservations strongly implicate Myo19 in starvation-induced

Fig. 3. Effect of cytoskeleton interfering drugs on Myo19–eGFP localization. Localization of Myo19–eGFP under (A, left) starvation conditions (St) in cellspre-treated with the actin-depolymerizing drug Latrunculin B (LatB) or EtOH (vehicle), or (B, left) following treatment with the microtubule inhibitor Nocodazole orDMSO (vehicle). Blue, nuclei; green, Myo19–eGFP; red, mitochondria. A quantification of foci localization of Myo19–eGFP is shown on the right. Myo19-expressing cells were scored according to the number of foci present under starvation in LatB or EtOH (vehicle) pre-treated cells, or upon the addition ofnocodazole or DMSO (vehicle). Values are presented as percentage of cells having the indicated foci number out of total analyzed cells (mean±s.d., n≥150 cellsfrom three independent experiments). Scale bars: 20 µm.

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filopodia formation. We next pursued the characterization of thephysical linkage between Myo19 and the mitochondria.

Endogenous Myo19 co-purifies with mitochondriaTo examine the nature of the physical interaction between Myo19and the mitochondria, we purified mitochondria fromHEK293 cellsusing differential centrifugation in order to biochemically verifyendogenous Myo19 subcellular localization. The differentialcentrifugation generates three crude fractions: a mitochondria-richfraction, called the heavy mitochondrial fraction (HMF), amitochondria-poor, called the light mitochondrial fraction (LMF),and a cytosolic fraction (CYT). Myo19 was present exclusively inthe HMF (Fig. 7A). To verify the mitochondrial localization, theHMF was further fractionated in a self-forming Iodixanol densitygradient. Mitochondria and ER co-fractionated to some extent(fraction 7–10), as seen by the existence of the mitochondrial andER markers VDAC and GRP94, respectively. Myo19 onlydistributed in the fractions of mitochondria and ER, but was

absent from the fraction of ER lacking mitochondria, indicating thatit interacts with the mitochondria (Fig. 7B). Notably, we did notdetect any dissociation of Myo19 throughout the rigorouspurification and fractionation procedure, suggesting that thisinteraction is stable.

Myo19 is anchored to the outer mitochondrial membraneWe next sought to determine the molecular basis underlying thephysical interaction of Myo19 with mitochondria. Severalpossibilities exist for protein–organelle interactions including sub-mitochondrial localization, interaction through a receptor ormediator protein or direct binding to the outer mitochondrialmembrane (OMM). The sub-mitochondrial localization of Myo19was determined by protease protection assay using proteinaseK. Myo19 was completely digested in the absence of any detergent,indicating that it resides on the outer mitochondrial membrane(OMM) (Fig. 7C, left). To test the membrane topology of Myo19,we purified mitochondria from Myo19–eGFP-expressing cells,

Fig. 4. Myo19–eGFP localizes to tips of filopodia in response to starvation. (A) Ectopic expression of Myo19–Halo and actin–eGFP reveals that Myo19–Halolocalizes to tips of actin protrusions in response to starvation. (B) Ectopic expression of Myo19–Halo and fascin–eGFP (top) or drf3/mDia2–eGFP (bottom)reveals that Myo19 localizes to the tips of growing filopodia in response to starvation. Mag., magnification of the white box. The intensity plot shows a colorintensity plot of the yellow line, generated using ImageJ. Blue, nuclei; green, actin–eGFP (A) fascin–eGFP (B, top) or drf3/mDia2 (B, bottom); Myo19–Halo stainedwith TMR. Scale bars: 20 µm (5 µm in magnification).

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Fig. 5. Myo19 knockdown prevents filopodia formation. (A) Ectopic expression of fascin–eGFP and subsequent starvation verify that the protrusions seen byDIC are indeed filopodia. Blue, nuclei; green, fascin–eGFP. Scale bars: 10 µm. (B) RNAi-mediated knockdown of endogenous Myo19 leads to a reduced proteinlevel expression by ∼80% compared to mock siRNA-treated cells. The ratio was calculated by dividing the intensity of Myo19 between the knockdown andthe mock treatment after each has been normalized according to actin. Band intensities were measured using ImageJ. (C) Images showing cells expressingfascin–eGFP under complete medium (left panel) or starvation medium (right panel) transfected with mock siRNA (upper row) or with Myo19 siRNA (lower row).The images show the strong effect on filopodia formation, length and numbers caused by Myo19 knockdown. (D) Quantification of filopodia number per cell (top)and filopodia length (bottom) from mock or Myo19-knockdown cells is given in box plots. The box represents the 25–75th percentiles. Small squares indicate themean, the median is indicated by a solid line through the box. Whiskers show the 10–90th percentiles. ‘X’ indicats the 1st and 99th percentiles, ‘-’ indicates maxand min values. Filopodia were measured from the base of the fascin-eGFP to its tip. n=500 filopodia from 36 cells from either mock or Myo19-knockdown cellsfrom three independent experiments for filopodia number per cell. n=900 filopodia from 60 cells from either mock or Myo19-knockdown cells from threeindependent experiments for filopodia length. ****P<0.0001 (one-tailed Student’s t-test). (E) Quantification of filopodia length distribution between WT cellstransfected with mock siRNA, Myo19 siRNA and Myo19 siRNA cells rescued with ectopic expression of Myo19–Halo. (F) Mitochondria were found in filopodia ofmock-treated cells in response to starvation (arrow). Scale bars: 10 µm. (G) Patches of fascin–eGFP could be seen at the cell periphery in response to starvationonly in Myo19 knockdown cells. Scale bars: 10 µm. (H) Mitochondria distribution was evaluated in Myo19-knockdown or mock-treated U2OS cells showingunchanged distribution of mitochondria. Blue, nuclei; red, mitochondria. Scale bars: 20 µm.

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where the eGFP tag is fused to the C-terminus of Myo19, andrepeated the protease protection assay. The proteinase-K-treatedsample was not recognized by anti-GFP antibody, indicating that theC-terminus faces the cytoplasm (Fig. 7C, right). ENDO G or ATPsynthase 5A (ATP5A), both mitochondrial intramembrane space(IMS) proteins, were used as a control to confirm that the IMS wasprotected from the protease. To address the possibility of protein–protein interactions betweenMyo19with an adaptor protein orOMMreceptor, we attempted to biochemically extract endogenous Myo19from purified mitochondria. The Myo19–mitochondria interactionwas resilient to high salt (2 M NaCl) and urea (2 M), indicating thatthe interaction is not limited to electrostatics or to protein–proteininteraction but that there is probably an additional binding feature. Asa control for the salt and urea extraction, we utilized ATP5A, whichcan be extracted by salt and urea when mitochondria are subjected tofreeze–thaw cycles (Fig. 7D). Interestingly, Myo19 could only beextracted by carbonate extraction from theOMM, suggesting that it isa peripheral membrane protein (Fujiki et al., 1982) (Fig. 7D).Furthermore, Myo19 separated to the detergent phase in a TritonX-114 phase separation of purified mitochondria, whereas thesoluble protein GRP94 was found in the aqueous phase (Fig. 7E).These findings suggest thatMyo19 is amonotopic membrane proteinthat does not transverse the OMM, with both the N- and C-terminusfacing the cytosol.

Residues 860–890 of Myo19mediate the interaction with theOMMTo explore the mode of Myo19–OMM interaction, we searched forputative membrane-binding motifs in Myo19 tail domain, which

has been shown to localize to mitochondria when ectopicallyexpressed (Quintero et al., 2009). Using the DAS predictionwebserver, we identified such a motif between amino acids 860and 890 (Cserzo et al.) (Fig. S4A). We ectopically expressedMyo19860–890–eGFP as the predicted membrane-binding motif andverified that this motif was sufficient to target eGFP to mitochondria(Fig. 8A). To test whether this domain is essential for Myo19 OMMlocalization, we ectopically expressed a deletion mutant lacking thisregion [Myo19824–970(Δ860–890)–eGFP]. This deletion mutant wasmostly distributed throughout the cytosol, although it was not fullyexcluded from mitochondria (compared to eGFP, Fig. S4B).Therefore, we generated a larger deletion, Myo19824–970(Δ851–895)–eGFP, which was further excluded from mitochondria but notcompletely. These results strongly support the hypothesis that theMyo19860–890 motif is both essential and sufficient for OMMlocalization; however, we cannot exclude the presence of a minorbinding region or other protein–protein region within the taildomain of Myo19 (Fig. 8A).

To test whether the Myo19860–890 membrane motif only dictatesOMM targeting or whether it also participates in Myo19 OMManchorage, we performed biochemical extractions on purifiedmitochondria from cells overexpressing Myo19860–890–eGFP.

In accordance to our finding in cells (Fig. 8A), Myo19860–890–eGFP was co-purified with mitochondria; however, unlike theendogenous Myo19, it appeared to be more sensitive to extractionby salt. Carbonate completely extracts Myo19860–890–eGFP frompurified mitochondria, similar to endogenous Myo19 (Fig. 8B). Thedifference in salt sensitivity might be due to an insufficient length ofthe motif meaning it is not fully anchored in the membranecompared to in the WT endogenous protein. Another possibility isthat a certain part of Myo19860–890–eGFP is correctly anchored,whereas the rest was present there nonspecifically (e.g. throughdimerization of eGFP). Nevertheless, we can conclude from theseexperiments that this motif is both essential and sufficient for OMMlocalization.

To further explore the OMM targeting, we examined the motifsequence. Although there is no consensus sequence for targetingproteins to the OMM, it has been shown that a moderatelyhydrophobic motif flanked by positive residues is required forOMM localization (Rapaport, 2003). Myo19 membrane motifcontains a moderately hydrophobic region between residues 864and 880, flanked by basic positively charged residues (Table S1,flanked by ‘*’). Therefore, we sought to assess whether Myo19OMM localization follows these criteria. Point mutations of thebasic residues, R882S and K883S, singly or in combination,resulted in a dramatic shift of the intracellular localization ofMyo19 to the ER (Fig. 8C; Table S1, Fig. S4C). Increasing thehydrophobicity of the hydrophobic region by the point mutationP865V resulted in dual localization to both the ER and themitochondria. Analysis of the P865V mutation shows that thehydrophobicity of the motif increased from 1.141 to 1.482 (Kyteand Doolittle, 1982) and, hence, this residue might participate indictating the correct mitochondrial localization of Myo19 (Fig. 8C;Table S1). Both results are in accordancewith similar studies on tail-anchored OMM proteins (Kanaji et al., 2000; Borgese et al., 2001,2007; Kaufmann et al., 2003; Wattenberg et al., 2007). In addition,the membrane motif with surrounding residues is predicted tocontain two α-helices at 856–878 and 882–892 according to theMemBrain webserver (Fig. S4D) (Yang et al., 2013). By plottingthis region as a helical wheel using HeliQuest (Gautier et al., 2008),one could observe that the N-terminal portion of the helix hasamphiphatic characteristics, which can be found in many

Fig. 6. Analysis of filopodia formation. (A) Myo19–Halo- and fascin–eGFP-expressing cells were starved and z-stack images were collected, revealingthat Myo19 localized to tips of peripheral filopodia but not dorsal filopodia.Scale bars: 20 µm (5 µm in maginifcation). (B) Analysis of the angle ofperipheral filopodia. Fascin–eGFP-expressing cells were starved and z-stackimages were taken. The peripheral filopodia angle was calculated using ImarisV8.0.0. (C) The rate of filopodia formation (mean±s.d.) was measured byperforming time-lapse imaging of starved U2OS cells expressing fascin–eGFPand transfected with mock or Myo19 siRNA and plotting the length of filopodiaover time. n=30 filopodia from each group, no statistical difference was foundusing a one-sided Student’s t-test.

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membrane-associated proteins (Hristova et al., 1999) (Fig. S4E).Collectively, these results show that theMyo19–OMM interaction ishighly specific and mutations in this motif dramatically disrupt thisinteraction.

Myo19-derived peptide binds to OMM-mimicking vesiclesTo characterize the Myo19 membrane-binding motif, we studiedthe binding of a synthetic peptide representing Myo19 residues851–895 (Table S2) to small unilamellar lipid vesicles (SUVs)with a phospholipid composition similar to the OMM [55%phosphatidylcholine, 30% phosphatidylethanolamine, 13%phosphatidylinositol and 2% dioleoyl phosphatidylserine (deKroon et al., 1997)]. Binding was followed by fluorescenceanisotropy, relying on two tryptophan residues that are present inthe peptide. The binding of Myo19 peptide to vesicles resulted inboth an increase in the anisotropy and enhanced fluorescence signal(Fig. 8D). Equilibrium binding of Myo19 peptide to vesicles wasmonitored by titrating peptide versus increasing vesicleconcentration. This resulted in a hyperbolic binding curve of boththe fluorescence anisotropy and the fluorescence total intensity(FTI) (Fig. 8D). Therefore, to account for the FTI change during thefluorescence anisotropy measurements, we applied global fitting toboth signals arising from peptide binding to vesicles to compensate

for the change in fluorescence yield during fluorescence anisotropymeasurements. Globally fitting the data to a hyperbolic bindingequation (see Eqn 3 in the Materials and Methods) yielded anapparent binding constant of KD=281±50 μM (mean±s.d., n=3). Toverify our results that endogenous Myo19 cannot be released fromthe OMM by high salt in vitro, we examined the salt-dependence ofthe binding interaction between the Myo19 peptide and the vesicles.As expected, equilibrium binding of Myo19 peptide to vesicles as afunction of [NaCl] up to 1 M exhibited no effect on the magnitudeof binding (Fig. 8E, inset, slope≈0), supporting our above findings(Fig. 7D). We performed additional binding experiments using ashorter peptide derived from Myo19 membrane motif that containsthe peak in predicted helix propensity and the corresponding ERmutant (Fig. S4D, Table S2, Myo19858-883 and Myo19858-RK882/3SS,respectively) to either OMM or ER mimicking vesicles [20%phosphatidylethanolamine, 66% phosphatidylcholine, 9%phosphatidylinositol, 3% dioleoyl phosphatidylserine (Watanabeet al., 1996)]. Both peptides showed similar affinities to OMM-mimicking vesicles (45.0±14.1 and 49.0±17.2 μM for WTor mutant peptide, respectively, Fig. 8F; mean±s.d., n=2). Thesmaller Myo19-derived peptide exhibited tighter affinity than thelonger one. This observation might reflect the predicted higherspecificity that resides in the shorter peptide than the longer peptide.

Fig. 7. Endogenous Myo19 is anchored to the OMM. (A) Fractionation of HEK293 cells using differential centrifugation to three main fractions; the heavymitochondrial fraction (HMF), which contains most of the mitochondria of the cell, the light mitochondrial fraction (LMF), which contains mostly ER and somemitochondria, and the cytosol (Cyt). Anti-VDAC, anti-Grp94 and anti-GAPDH antibodies were used as markers for mitochondria, ER and cytosol, respectively.(B) Fractionation of the HMF using a self-forming Iodixanol gradient (OptiPrep). Fractions of a fixed volume were collected with a pipette from the top of thegradient following the centrifugation step. VDAC and GRP94 were used as markers for mitochondria and ER, respectively. (C) Protease protection assay onpurified mitochondria from WT cells (left) or cells ectopically expressing Myo19–eGFP (right) using proteinase K (PK). Anti-Myo19 targets the Myo19 N-terminalregion. ENDO G and ATP synthase 5A (ATP5A) were used as markers for the intermembrane space, indicating that the OMM was left intact. VDAC is an OMMintegral protein. (D) Extraction of membrane proteins using high salt, urea or carbonate. Purified mitochondria were treated as indicated in the Materials andMethods, and then membranes were separated by centrifugation and resolved by SDS-PAGE. Pel and Sup represent the pellet and supernatant, respectively.(E) Detergent extraction by Triton X-114. Purified mitochondria were lysed, incubated at 37°C for 3 min to form micelles and pelleted at 300 g for 3 min.The aqueous (Aq) and detergent (Det) phases were collected separately. VDAC and GRP94 were used as control for membrane and soluble proteins,respectively.

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Fig. 8. Myo19 membrane motif. (A) Colocalization of Myo19860–890–eGFP, Myo19824–970(Δ860–890)–eGFP or Myo19824–970(Δ851-895)–eGFP with mitochondria.The right panel is a magnification of the white boxes from the left panel. The white arrow emphasizes lack of colocalization. Blue, nuclei; green, eGFP-fusedMyo19; red, mitochondria. Scale bars: 20 μm (left panels), 10 μm (right panels). (B) Extraction of membrane proteins using high salt or carbonate. Mitochondriawere purified from Myo19860–890–eGFP expressing cells and treated as indicated in the Materials and Methods. Membranes were separated by centrifugationand resolved on SDS-PAGE. Pel and Sup represent the pellet and supernatant, respectively. (C) Colocalization of Myo19824–970–eGFP and mutants withmitochondria or ER. Blue, nuclei; cyan, ER (SRβ–CFP); green, eGFP-fused Myo19; red, mitochondria. Scale bars: 20 µm. (D) Equilibrium binding of Myo19peptide to SUVs. Peptide containing the motif essential for OMM interaction (Myo19851–895) was assayed for binding to vesicles by fluorescence anisotropy.Titrating vesicles versus peptide exhibits a hyperbolic dependence-binding curve. Black and green squares mark the fluorescence anisotropy and the totalfluorescence intensity (FTI), respectively. Black and green lines are the globally fitted curves using Eqn 3 for the fluorescence anisotropy and FTI data,respectively. Peptide was used at 10 µM. Results are mean±s.d. (n=3). (E) Equilibrium binding of Myo19851–895 peptide to vesicles as a function of increasing[NaCl]. The binding of the peptide to the vesicles was unaffected by increasing [NaCl], demonstrating an unchanged slope. Peptide was used at 10 µM,and vesicles concentration was 150 µM. Results are mean±s.d. (n=2). (F) Equilibrium binding of Myo19 and Zeta1 peptides to SUVs. Peptides containing theshorter peptide derived from Myo19 membrane motif that contains the peak in predicted helix propensity (Myo19858-883), the corresponding ER mutant(Myo19858–883RK882/3SS) and a hydrophilic peptide (Zeta1) were assayed for binding to ER or OMM-mimicking vesicles by fluorescence anisotropy.Titrating vesicles versus peptide exhibits a hyperbolic dependence-binding curves. Lines are the globally fitted curves using Eqn 3 for the fluorescenceanisotropy and FTI. Peptide concentrations were 4 µM. Results are mean±s.d. (n=2).

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The WT peptide had also a similar affinity to ER-mimickingvesicles (50.5±22.0 μM), not showing evident preference forOMM-mimicking vesicles. In vitro binding of a hydrophilicpeptide (Zeta1, Table S2), was much weaker (∼2.5 fold)compared to the Myo19 peptide (124.4±18.7 μM), supporting thenotion that Myo19–OMM binding is mediated by hydrophobicinteractions (Fig. 8F). Although our newly identified membranemotif is essential and sufficient for OMM localization in cells, thecellular specificity might be achieved by unique, yet unidentified,cytosolic or mitochondrial components.

DISCUSSIONMitochondria are a key regulator of many stress conditions that cellsmight encounter, such as deprivation of various nutrients. Tosurvive, cells must possess mechanisms that enable them to regainhomeostasis. We therefore focused our investigation on the functionof Myo19 in the cellular response to stress by glucose starvation, amajor metabolite that is key to many cellular signaling pathways anda stress condition that occurs in solid tumors (Acker and Plate, 2002;Schroeder et al., 2005; Tredan et al., 2007; Hirayama et al., 2009;Moruno et al., 2012). We demonstrate a new function for Myo19 infilopodia formation and mitochondria localization under the cellularglucose-starvation stress response.Glucose starvation of U2OS cells ectopically expressing Myo19

resulted in striking localization of Myo19 and mitochondria totips of newly formed starvation-induced filopodia in a motor-dependent manner. Interestingly, these experiments demonstrated adirect link between active Myo19 and the actin cytoskeleton fortheir localization to the tips of starvation-induced filopodia.Knockdown of Myo19 by siRNA revealed its contribution to theformation and elongation of these filopodia, strongly supporting thehypothesis that Myo19 plays a role in starvation-induced filopodiaformation. Finally, we show the molecular details of Myo19–mitochondria interactions, which is achieved through a unique∼30–45-residue motif that enables it to be directly anchored to theOMM. To the best of our knowledge, this is the first myosin that isanchored in the membrane, in contrast to other myosins, whichinteract with phospholipids through electrostatic interactions(Hokanson and Ostap, 2006; Spudich et al., 2007). Thisdistinctive feature most likely facilitates strong force bearingability, preventing detachment of Myo19 from the mitochondriaand overcoming high drag forces and competing interactions.Filopodia are actin-based protrusions that extend from the

lamellipodia and have been shown to be important in cancer cellmigration, metastasis and angiogenesis (Folkman, 2002; Machesky,2008; Shibue et al., 2012). Filopodia formation depends primarilyon ATP-actin polymerization at the growing tip, providing theenergy to push the membrane forward (Mallavarapu and Mitchison,1999). Molecular modeling performed by Papoian and coworkers(Lan and Papoian, 2008) has modeled the rate of filopodia growth asbeing dependent on three factors: the resistance of the cellmembrane, the mechanical buckling forces that lead to breakageof filopodia filaments, and the transport of ATP-actin monomers tothe growing tips. The model also predicts a length-dependence ofATP-actin diffusion from the lamellipodium to the filopodia tips,where eventually the polymerization of actin will be limited byATP-actin concentration. Ectopically expressed class III myosinshave been shown to localize to tips of stereocilia, transporting espin-1, which results in a boost of their growth (Salles et al., 2009;Merrittet al., 2012). Myosin X has been found to undergo intrafilopodialmotility and function in filopodia formation (Berg and Cheney,2002; Tokuo and Ikebe, 2004; Bohil et al., 2006).

We propose that Myo19 localization to the fast growing ends ofthe filopodia promote their growth probably by mediating thelocalization of mitochondria to supply the high local ATP demandfor ATP-actin polymerization. Here, we noticed that not allfilopodia (<20%) contain mitochondria, and we speculate thatthere are small mitochondria at the filopodia tips, which are belowour detection limit. Another possibility is that not all filopodia arebeing actively elongated or fail to continue to mature. In support ofthis, it has been shown that mitochondria stalling at the axonbranching sites are necessary for efficient branching, an actin-polymerization-dependent process (Spillane et al., 2013). However,we cannot exclude a role for mitochondria in filopodial Ca2+

regulation (Davenport et al., 1996) or other signaling events leadingto filopodia formation.

Glucose starvation increases the cellular AMP:ATP ratio,triggering the activation of AMP-activated protein kinase(AMPK) to meet the energetic demand of the cell (Hardie et al.,2012). We found that the minimal glucose concentration thatprevents U2OS starvation-induced filopodia is extremely low.Complete medium contains 25 mM glucose, whereassupplementation of the starvation medium with as little as 5 µMprevents formation of starvation-induced filopodia (Table S3). Thisconcentration is much lower than necessary for glycolysis (Faulknerand Jones, 1978); therefore, we propose that the prevention ofMyo19–mitochondria localization to starvation-induced filopodia ismediated by glucose signaling events rather than metabolichomeostasis. In support of this, glucose starvation results inactivation of a unique signature of phosphotyrosine signalingassociated with focal adhesions, actin remodeling and F-actinstabilization (Arber et al., 1998; Miki et al., 1998; Graham et al.,2012). Glucose starvation increases reactive oxygen species (ROS)production (Diaz et al., 2009; Weaver, 2009; Graham et al., 2012;Taulet et al., 2012; Olguin-Albuerne and Moran, 2015). ROS havebeen shown to be essential for the formation of actin protrusions andwell-characterized invadopodia, which have been linked toincreased cancer invasiveness (Diaz et al., 2009; Weaver, 2009;Graham et al., 2012; Taulet et al., 2012; Olguin-Albuerne andMoran, 2015). Therefore, it could well be that the effect of Myo19on starvation-induced filopodia formation is regulated throughROS. It will be exciting to study whether glucose starvation andoxidative stress induces Myo19 post-transcriptional modifications(PTMs), such as phosphorylation, that might modulate its motoractivity. Here, we found that, under complete medium growthconditions, ectopic expression of Myo19 causes mitochondria toform globular structures. Previous reports have indicated thatectopic expression of Myo19 affects mitochondrial morphology,causing single mitochondria to obtain tadpole morphology;however, such globular structures have not previously beenreported (Quintero et al., 2009). Our analysis shows that theseglobular structures are in fact clumped mitochondria, rather thanhyperfused mitochondria; otherwise loss of MitoTracker intensitywould be throughout the entire globular structure (Fig. S1E). Thisindicates that Myo19 does not promote fusion of mitochondria;however its effect on mitochondrial fission and fusion has not yetbeen determined. Myo19 knockdown did not cause any obviousmorphological changes in the tubular mitochondria network. Thisinclines that Myo19 is not essential for tubular networkmorphology; however, we cannot exclude that it might also playsome role in intrinsic mitochondrial dynamics or that the reducedlevels are still sufficient to maintain the mitochondrial morphology.Corollary to our findings, knockdown of Myo19 has been shown tobe important for correct segregation of mitochondria in a subset of

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dividing cells, whereas the rest were correctly dividing (Rohnet al., 2014).The nature of Myo19 interaction with the mitochondria is

essential for understanding the linkage between mitochondria,Myo19 and the actin cytoskeleton. We demonstrate that Myo19residues 860–890 are essential and sufficient to cause it to localizeto the OMM in cells. This region is moderately hydrophobic, withflanking positive residues, which are essential for correctmitochondrial localization. This targeting system has been shownin several tail-anchored proteins. Tail-anchored proteins have amembrane-targeting domain in their last 30 residues, which isnested in the ribosome when the protein completes its translation,whereas the Myo19 membrane-targeting domain is followed by∼80 residues. The precise targeting mechanism of Myo19 to theOMM still remains to be determined. Myo19 might competebetween the ER and OMM membranes, between targeting proteinssuch as Asna1/TRC40 and a yet unidentified OMM targetingprotein, or transported through the ER to the OMM. Myo19membrane interaction is highly stable as deduced from ourbiochemical extractions of endogenous Myo19 and from ourbinding experiments of the OMM binding motif to vesicles. Inaddition to the increase in fluorescence anisotropy, we observedstrong tryptophan fluorescence increase upon binding of peptidecontaining the OMM-binding motif to vesicles. Tryptophanfluorescence increase is associated with a shift of the residue froma hydrophilic to hydrophobic environment, suggesting some degreeof membrane insertion of the OMM-binding motif (Tsai, 2006). Wepropose with high likelihood that Myo19 is permanently bound tothe OMM through its entire lifetime. This implicates that theregulation of Myo19 activity is most likely performed by amechanism that modulates its ATPase activity while it is anchoredto the OMM. It will be fascinating in the future to study themechanism by which Myo19 localizes to the OMM from themoment of its translation as it anchors to the membrane. Myo19enzymatic adaptation has not yet been characterized. It will becrucial to study its mechanochemical properties and how they relateto its cellular function (De La Cruz and Ostap, 2004; Geeves et al.,2005; Henn and Sadot, 2014).Myo19 has recently been implicated in gliomagenesis (Jalali

et al., 2015). Moreover, mitochondria were found in filopodia ofglioblastoma cells that actively penetrate extracellular matrix(Arismendi-Morillo et al., 2012). Taken together with ourobservation that knockdown of Myo19 attenuates filopodiaformation without evident effects on the mitochondrial networkorganization, we propose that Myo19 might be a therapeuticcandidate for cancer therapy.

MATERIALS AND METHODSReagentsAll chemicals, reagents and media were purchased from Sigma (St Louis,MO) unless otherwise indicated.

Myo19 cloning and plasmidsMyo19 was cloned from an EST library (NIH clone ID 3629870) cDNA.The amplified PCR product was missing the region encompassingnucleotides 1318–1916 (database sequence FLJ22865). Therefore, thisregion was artificially synthesized and cloned into pFC14K (Promega).Full-length Myo19 and its tail domain (residues 824–970) weresubcloned to peGFP-N1b using restriction enzymes and used as atemplate for truncations and mutagenesis. Table S4 contains the primersused for the cloning. peGFP-N1 was a gift from Nabieh Ayoub(Technion Israel Institute of Technology, Haifa, Isreal), fascin–eGFP was

a gift from Alexander Bershadsky (Weizmann Institute of Science,Rehovot, Isreal). mEmerald–Vinculin-N-21 was a gift from MichaelDavidson (Addgene plasmid number 54304) (Burnette et al., 2011).pRK-GFP-Paxillin was a gift from Kenneth Yamada (Addgene plasmidnumber 50529). eGFP-tagged full-length mDia2/Drf3 was a kind giftfrom Klemens Rottner and Jan Faix (TU Braunschweig and HanoverMedical School, Germany, respectively).

Cell culture, cell lines, starvation conditions and cytoskeletoninterfering drugsU2OS cells (gift of Nabieh Ayoub) were grown at 37°C and under 5%CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 10% fetal calf serum (FCS, Biological Industries, Beit Haemk,Israel), 4.5 g/l glucose, 2 mM L-Glutamine, 20 mM HEPES-KOH pH 7.4,100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericinB. HEK293SF-3F6 (ATCC) for purification of mitochondria and cellorganelles were grown in suspension in EX-CELL 293 (Sigma) at 37°Cand 5% CO2. For starvation conditions, cells were rinsed twice with PBSand incubated in starvation medium (glucose-free DMEM supplementedwith 20 mM HEPES-KOH pH 7.4, 5 mg/ml BSA, 100 U/ml penicillin,100 µg/ml streptomycin and 0.25 µg/ml amphotericin B) for 2 h. Drugsthat interfered with the cytoskeleton (final concentration: 0.2 µMLatrunculin B or 0.75 µg/ml nocodazole) were first diluted in mediumand then added to the cells. Cells were tested for mycoplasmacontamination and found clean (EZ-PCR mycoplasma detection kit,Biological Industries).

Purification of mitochondriaHEK293SF-3F6 cells were harvested by centrifugation at 200 g, washedtwice with PBS and once in homogenization medium (0.25 M sucrose,1 mM EDTA, 20 mM HEPES pH 7.4). The pellet was resuspended inhomogenization medium containing protease inhibitors (0.1 mMbenzamidine, 0.055 mM phenanthroline, 1 mM PMSF and, from AGScientific, 0.01 mM bestatin, 0.02 mM leupeptin and 0.005 mM pepstatinA), homogenized using a teflon-glass pestle and centrifuged at 1000 g topellet nuclei. The resulting post-nuclear supernatant was centrifuged for20 min at 10,000 g to pellet mitochondria and obtain a heavy mitochondrialfraction (HMF). The supernatant from the 10,000 g centrifugation was takenand centrifuged for 20 min at 17,000 g to pellet light membranes and obtaina light mitochondrial fraction (LMF). The supernatant from the 17,000 gwas taken to obtain a cytosolic fraction (CYT).

Additional purification of mitochondriaTo obtain pure mitochondria we used isopycnic ultracentrifugation. TheHMF was resuspended in homogenization medium containing proteaseinhibitors and adjusted to 17.5% OptiPrep. The HMF was centrifuged at270,000 g for 3 h in a Sorvall T-890 or Beckman Type 70 rotor to form aself-forming density gradient. Following ultracentrifugation, three bandsappeared: light membranes, mitochondria and a DNA-rich band (from topto bottom). The second band was taken using a pasture pipette, diluted 1:1with homogenization medium containing protease inhibitors and centrifugedfor 30 min at 17,000 g to pellet mitochondria. The mitochondria wereresuspended in fresh homogenization medium containing proteaseinhibitors at ∼5–6 mg/ml, concentration was measured using a Bradfordassay according to the manufacturer’s directions (BioRad). Purifiedmitochondria were flash frozen in liquid nitrogen and stored at −80°C.All steps were performed at 4°C.

Protease protection assayFor proteinase K digestion, mitochondria were washed three times withhomogenization medium to remove protease inhibitors. Proteinase K wasadded at 50 µg/ml and incubated on ice for 30 min. Reactions wereterminated by addition of PMSF to 2 mM and centrifugation at 10,000 g for3 min to pellet mitochondria and to remove the protease. Sample buffer ×5(final concentration, 50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1%β-mercaptoethanol, 0.02% Bromophenol Blue) was added to the samplesand they were resolved by SDS-PAGE.

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Membrane extractionPurified mitochondria were resuspended in either 100 mMNaCO3 pH 11.5,2 M NaCl in homogenization medium, or 2 M urea in 20 mMMES pH 6.5for 30 min on ice, during which the NaCl and urea samples were subjectedto ten freeze–thaw cycles in liquid nitrogen to mechanically break themitochondria. The samples were then centrifuged for 1 h at 150,000 g in aSorvall S100-AT3 rotor. The supernatant was collected and the pellet wasresuspended in an equal volume of 100 mM HEPES pH 7.4. To precipitatethe extracted proteins, we performed trichloroacetic acid (TCA) and acetoneprecipitation on the NaCO3 and NaCl supernatant, TCA was added to 12%from a 100% stock and allowed to incubate for 1 h at 4°C. The samples werecentrifuged for 30 min at 17,000 g, resuspended with ice-cold acetone andleft overnight at −20°C. The samples were then centrifuged for 30 min at17,000 g, supernatant was removed and the acetone was allowed toevaporate by heating the samples for 10 min at 90°C. Urea-containingsamples were desalted using Zeba spin desalting columns 7K MWCOaccording to the manufacturer’s protocol (Thermo Fisher Scientific).Sample buffer ×5 (as described in the protease protection assay section)was added to the samples and they were resolved by SDS-PAGE.

Triton X-114 extractionPurified mitochondria were resuspended in cold phase separation buffer[10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-114 (MPBiomedicals); protease inhibitors as described in the purification ofmitochondria section] and incubated on ice for 5 min, insoluble materialwas removed by centrifugation at 10,000 g for 10 min. The lysate wasloaded on top of a cold sucrose cushion (10 mM Tris-HCl pH 7.4,150 mM NaCl, 0.06% Triton X-114, 6% sucrose, protease inhibitors),incubated at 37°C for 3 min and then centrifuged at 300 g for 3 min. Theupper phase was placed in a new tube and Triton X-114 was added to0.5%; the solution was then incubated on ice until it cleared, loaded on thesucrose cushion, incubated at 37°C for 3 min and then centrifuged at 300 gfor 3 min. This process was repeated again with the exception that TritonX-114 was added to 2%. The upper and the lower phase were collectedand corrected to the same volume. Sample buffer ×5 (described in theprotease protection assay section) was added to the samples and they wereresolved by SDS-PAGE.

Western blottingFollowing SDS-PAGE, gels were washed in Milli-Q water. Nitrocellulose orPVDF membranes (PVDF membranes were hydrated in 100% methanol for 1min) were incubated for 1 min in transfer buffer and transfer was performed for10 min using a semi-dry blotter (GenScript). Transferred membranes werewashed with Milli-Q water and blocked using EZblock (Biological Industries)for 1 h at room temperature. Following blocking, primary antibody was addedfor 1 h at room temperature or left overnight at 4°C [0.5–1 µg/ml anti-VDACantibody abcam ab14734, anti-Myo19 antibody Sigma HPA021415 or Abcamab174286, anti-GRP94 antibody (a kind gift from Christopher Nicchitta,Department of Cell Biology and Biochemistry, Duke University, NorthCarolina), anti-HSP60 antibody (Santa Cruz Biotechnology sc-59567), anti-ENDOGantibody (SantaCruzBiotechnologysc26923), anti-ATP5Aantibody(abcam ab14748) anti-GFP antibody (Roche 11814460001)]. The membranewaswashedwith TBS-T (TBS, 50 mMTris pH 7.5, 150 mMNaClwith 0.02%Tween-20) and horseradish peroxidase (HRP)-conjugated secondary antibodywas added for 1 h, washed with TBS-T and developed using 0.01% H2O2,1.25 mM Luminol and 0.04 mM p-Cumaric acid in 0.1 M Tris-HCl pH 8.5.Images were acquired using a ImageQuant LAS4000 imager (GE).

Transfections and microscopyTransfections were performed using polyethylenimine (PEI, WeisScientific-PolySciences). Adherent U2OS cells were plated a day beforetransfection on plastic or glass bottom dishes and allowed to adhereovernight. Plasmid DNA and PEI were diluted separately in 150 mM NaCland then combined, and complex formation was allowed to occur for 25 minat room temperature before addition to the cells and incubation overnight.Hoechst 33342 (0.75 µg/ml), MitoTracker (30 nM, Molecular Probes) andpropidium iodide (1 µg/ml) were added 15 min prior to imaging. HaloTag

constructs were stained by incubating the cells with HaloTag TMR LigandON (25 nM, Promega). Cells were imaged using Confocal Zeiss LSM 700or Confocal Zeiss LSM 710 or GE InCell Analyzer 2000 at ×63magnification in an environmental chamber.

ImmunofluorescenceCells grown on coverslips werewashed with PBS, and fixed with 4% PFA inPBS (EMS) at room temperature for 15 min. The PFA was removed bywashing with PBS, and the cells were blocked for 1 h with EZblock(Biological Industries). To visualize actin and nuclei, the cells wereincubated with Alexa-Fluor-488–phalloidin and Hoechst 33342 for 30 min.The cells were then washed with TBS and mounted on slides usingFluoromount-G.

RNA interferenceTransfections were performed using Lipofectamine 3000 (LifeTechnologies). Adherent U2OS cells were plated at 70–90%confluence a day before transfection on plastic dishes and allowed toadhere overnight. Mock siRNA (Universal negative control siRNA, IDT)or mix of three Myo19 siRNAs (10 nM each, reference numbers118568291, 118568285, 118568288, IDT), and Lipofectamine wereseparately resuspended in OptiMEM (Life Technologies). The RNAi mixwas added to the Lipofectamine mix and incubated for 5 min at roomtemperature. The medium was replaced with DMEM without serum andthe transfection mix was added. Complete medium was added after 4 h.On the next day, the cells were trypsinized and plated on glass bottomfluoridish (WPI) or optic bottom 24-well plates, for microscopy, orplastic dishes, for western blotting at 72 h post transfection.

Prediction webservers used in this studyThe webservers can be found at http://heliquest.ipmc.cnrs.fr/, http://www.csbio.sjtu.edu.cn/bioinf/MemBrain/, http://www.sbc.su.se/~miklos/DAS/.

Image analysis and calculation of mitochondria-positive Myo19–eGFP fociWestern blot band intensity, filopodia length measurements and line plotanalysis were performed using ImageJ (NIH). Filopodia anglemeasurements were performed using IMARIS V8.0.0 (Bitplane AG,Zurich, Switzerland).

To calculate the fraction ( f ) of mitochondria positive Myo19-eGFP foci(Fd) we calculated the ratio between the number of MitoTracker-Red-positive Myo19–eGFP foci (NFd) to the total number of Myo19–eGFP foci(NFt) according to the equation:

f ðFdÞ ¼ NFd

NFt:

Determination of filopodia growth rateTo determine the filopodia growth rate from the time-lapse imaging, we tookinto consideration only the region where the filopodia growth was constantover time (i.e. between 80 and 110 min). We used the following equation tocalculate individual filopodia growth rate vf :

vf ¼ l110 � l80Dt

;

where l110 is filopodia length at the time of 110 min, l80 is filopodia length atthe time of 80 min and elapsed time (Δt) is 30 min. N=30 for each treatment.

SUV preparationPhosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol anddioleoyl phosphatidylserine (Avanti) were mixed in chloroform andevaporated under nitrogen. The vesicles were then resuspended inbinding buffer (40 mM HEPES pH 7.1, 150 mM NaCl), subjected to tenfreeze–thaw cycles in liquid nitrogen and kept at −80°C. The vesicleswere thawed and sonicated for 5 min at low intensity; insoluble material

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was pelleted by centrifugation at 100,000 g for 20 min. Vesicles wereused within 2 days after sonication.

Microirradiation of mitochondriaU2OS cells were loaded with 25 nM of the MitoTracker dye (sensitive tomitochondrial membrane potential) for 15 min at 37°C. Unbound dye wasremoved by three washes with complete medium. A 0.5×0.5 µm square wasirradiated using a 405 nm laser at 100% intensity, causing singlemitochondria to depolarize and lose staining.

Fluorescence anisotropy measurements and KD determinationEquilibrium binding by fluorescence anisotropy measurements wasperformed with a PC1 spectrofluorimeter (ISS, Champaign, IL) designedas a T-format for simultaneous acquisition on two emission channelmonochromators equipped with automatic polarizers. Samples wereequilibrated (60 min, room temperature) and then measurements wereperformed by following intrinsic fluorescence of the peptide withλex=280 nm using vertical polarized light, and the emitted vertical andhorizontal polarized light was monitored at 90° with double emissionmonochromators at λem=335 nm. The G-factor for correction of the differentgain between the dual PMT detectors was calculated as described by theinstrument manufacturer. The fluorescence total intensity (FTI) and totalfluorescence anisotropy are given by:

FTI ¼ ðGIk þ 2I?Þ ð1Þand

Fluorescence anisotropy ¼ ðGIk � I?ÞðGIk þ 2I?Þ ; ð2Þ

where the I|| is the parallel fluorescence intensity and I⊥ is the perpendicularfluorescence intensity.

Our binding model for a simple bimolecular reaction is:

P þ VNKDPV ; ¼ ½P�½V �

½PV � :

Under the condition of Ptot≪KD then the general solution for thisequilibrium binding scheme is in the form of the following quadraticequation:

½PV � ¼ðPtot þ Vtot þ KDÞ �

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðPtot þ Vtot þ KDÞ2 � 4PtotVtot

q

2; ð3Þ

where Ptot is monitoring species; Vtot is titrating species and [PV] is thebound species.

The total fluorescence and anisotropy were fitted globally (Origin Lab9.0) according to Otto et al. (1994) and Henn et al. (2010).

Statistical analysisStatistical significance between mock and treated groups was performedusing Student’s t-test, the different groups were considered to have the samevariance because the groups were taken from the same pool of cells.Statistical significance was scored as *P≤0.05, **P≤0.01, ***P≤0.001,****P≤0.001. All presented error bars indicate standard deviation.

AcknowledgementsWe acknowledge Dr Dan Cassel for his assistance in the vesicle preparation, andcritically reading the manuscript. We thank Drs Yehuda G. Assaraf and NabiehAyoub for their invaluable comments on the manuscript. We are grateful forDr Nitzan Dahan’s assistance with the fluorescence microscopy imaging from LifeSciences and Engineering Infrastructure Unit at the Technion.We thank DrsMichaelDavidson and Kenneth Yamada for providing the focal adhesion marker plasmids.We thank Drs Klemens Rottner and Jan Faix (TU Braunschweig and HanoverMedical School, Germany, respectively) for providing the eGFP-tagged full-lengthmDia2/Drf3 plasmid.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsB.I.S. designed and carried out experiments, analyzed the data and co-wrote themanuscript. M.U. designed and carried out experiments and analyzed the data.A.H. supervised the project, designed and analyzed the data and co-wrotethe manuscript.

FundingM.U. is supported by the Lady Davis Fellowship and Israeli Government Ministry ofForeign Affairs Scholarship. A.H. acknowledges the Marie Curie Career Integration[grant number 1403705/11].

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.175349/-/DC1

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