Mitochondria-associated myosin 19 processively transports mitochondria on actin tracks in living cells
2022; Elsevier BV; Volume: 298; Issue: 5 Linguagem: Inglês
10.1016/j.jbc.2022.101883
ISSN1083-351X
AutoresOsamu Satō, Tsuyoshi Sakai, Young-Yeon Choo, Reiko Ikebe, Tomonobu M. Watanabe, Mitsuo Ikebe,
Tópico(s)Microtubule and mitosis dynamics
ResumoMitochondria are fundamentally important in cell function, and their malfunction can cause the development of cancer, cardiovascular disease, and neuronal disorders. Myosin 19 (Myo19) shows discrete localization with mitochondria and is thought to play an important role in mitochondrial dynamics and function; however, the function of Myo19 in mitochondrial dynamics at the cellular and molecular levels is poorly understood. Critical missing information is whether Myo19 is a processive motor that is suitable for transportation of mitochondria. Here, we show for the first time that single Myo19 molecules processively move on actin filaments and can transport mitochondria in cells. We demonstrate that Myo19 dimers having a leucine zipper processively moved on cellular actin tracks in demembraned cells with a velocity of 50 to 60 nm/s and a run length of ∼0.4 μm, similar to the movement of isolated mitochondria from Myo19 dimer-transfected cells on actin tracks, suggesting that the Myo19 dimer can transport mitochondria. Furthermore, we show single molecules of Myo19 dimers processively moved on single actin filaments with a large step size of ∼34 nm. Importantly, WT Myo19 single molecules without the leucine zipper processively move in filopodia in living cells similar to Myo19 dimers, whereas deletion of the tail domain abolished such active movement. These results suggest that Myo19 can processively move on actin filaments when two Myo19 monomers form a dimer, presumably as a result of tail–tail association. In conclusion, Myo19 molecules can directly transport mitochondria on actin tracks within living cells. Mitochondria are fundamentally important in cell function, and their malfunction can cause the development of cancer, cardiovascular disease, and neuronal disorders. Myosin 19 (Myo19) shows discrete localization with mitochondria and is thought to play an important role in mitochondrial dynamics and function; however, the function of Myo19 in mitochondrial dynamics at the cellular and molecular levels is poorly understood. Critical missing information is whether Myo19 is a processive motor that is suitable for transportation of mitochondria. Here, we show for the first time that single Myo19 molecules processively move on actin filaments and can transport mitochondria in cells. We demonstrate that Myo19 dimers having a leucine zipper processively moved on cellular actin tracks in demembraned cells with a velocity of 50 to 60 nm/s and a run length of ∼0.4 μm, similar to the movement of isolated mitochondria from Myo19 dimer-transfected cells on actin tracks, suggesting that the Myo19 dimer can transport mitochondria. Furthermore, we show single molecules of Myo19 dimers processively moved on single actin filaments with a large step size of ∼34 nm. Importantly, WT Myo19 single molecules without the leucine zipper processively move in filopodia in living cells similar to Myo19 dimers, whereas deletion of the tail domain abolished such active movement. These results suggest that Myo19 can processively move on actin filaments when two Myo19 monomers form a dimer, presumably as a result of tail–tail association. In conclusion, Myo19 molecules can directly transport mitochondria on actin tracks within living cells. Myosins are motor proteins that play critical roles in various cell functions, such as force production, cell motility, morphosis, cytokinesis, vesicle, or macromolecule transportation, and organelle localization through association with actin filaments (1Coluccio L.M. Myosins: A Superfamily of Molecular Motors. Springer, Dordrecht2008Crossref Google Scholar). A number of types of myosins have been discovered, and based on the primary structure of their motor domain, it is thought that >35 classes of myosins are constituted of the superfamily (2Odronitz F. Kollmar M. Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species.Genome Biol. 2007; 8: R196Crossref PubMed Scopus (291) Google Scholar). All myosins contain a conserved motor domain and neck domain, followed by a tail domain specific to the myosin class. The motor domain is responsible for actin binding and converting the chemical energy of ATP to mechanical work. Following short neck domain containing IQ motifs interacts with calmodulin (CaM) and/or CaM-like light chain(s) and constitutes a lever arm when myosin moves on actin filaments. In addition, this domain also functions as a regulatory domain of various myosins. A C-terminal tail domain is structurally unique and is often critical for the class-specific functions, such as regulation of the motile activity, association with unique binding partners, and targeting the molecule to the specific intracellular structures. Several myosins in the myosin superfamily members have a coiled-coil domain after the neck region to facilitate its dimer formation, whereas others do not have, and it is thought that the dimer formation of myosin is important for the function and regulation of myosin molecules (3Yu C. Feng W. Wei Z. Miyanoiri Y. Wen W. Zhao Y. Zhang M. Myosin VI undergoes cargo-mediated dimerization.Cell. 2009; 138: 537-548Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 4Umeki N. Jung H.S. Sakai T. Sato O. Ikebe R. Ikebe M. Phospholipid-dependent regulation of the motor activity of myosin X.Nat. Struct. Mol. Biol. 2011; 18: 783-788Crossref PubMed Scopus (78) Google Scholar, 5Sakai T. Umeki N. Ikebe R. Ikebe M. Cargo binding activates myosin VIIA motor function in cells.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 7028-7033Crossref PubMed Scopus (54) Google Scholar, 6Quintero O.A. Yengo C.M. Myosin X dimerization and its impact on cellular functions.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 17313-17314Crossref PubMed Scopus (4) Google Scholar). Recent studies have revealed that several types of myosin can continuously move on actin filament, which are thought to be involved in cargo transportation in cells (7Ross J.L. Ali M.Y. Warshaw D.M. Cargo transport: Molecular motors navigate a complex cytoskeleton.Curr. Opin. Cell Biol. 2008; 20: 41-47Crossref PubMed Scopus (260) Google Scholar). These myosins are often called processive myosin, and it is thought that these myosins spend a majority of the actin-activated ATP hydrolysis cycle time in the "strong actin-binding" state called high duty cycle myosin (8De La Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. The kinetic mechanism of myosin V.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13726-13731Crossref PubMed Scopus (361) Google Scholar). It is thought that two-headed myosins having a high duty cycle can processively walk on actin filaments with hand-over-hand mechanism (9Yildiz A. Forkey J.N. McKinney S.A. Ha T. Goldman Y.E. Selvin P.R. Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization.Science. 2003; 300: 2061-2065Crossref PubMed Scopus (1586) Google Scholar). Myo19 (also called myosin 19 or class XIX myosin) is an unconventional myosin that is expressed in vertebrates. From the primary structure, it is predicted that Myo19 consists of a motor domain, a neck domain containing three IQ motifs, and a unique tail domain, but missing a predicted coiled-coil domain (2Odronitz F. Kollmar M. Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species.Genome Biol. 2007; 8: R196Crossref PubMed Scopus (291) Google Scholar, 10Berg J.S. Powell B.C. Cheney R.E. A millennial myosin census.Mol. Biol. Cell. 2001; 12: 780-794Crossref PubMed Scopus (628) Google Scholar). It has been reported that Myo19 is colocalized with mitochondria (11Quintero O.A. DiVito M.M. Adikes R.C. Kortan M.B. Case L.B. Lier A.J. Panaretos N.S. Slater S.Q. Rengarajan M. Feliu M. Cheney R.E. Human Myo19 is a novel myosin that associates with mitochondria.Curr. Biol. 2009; 19: 2008-2013Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) and has been considered to play a role in actin-based mitochondria dynamics in cells (12Rohn J.L. Patel J.V. Neumann B. Bulkescher J. McHedlishvili N. McMullan R.C. Quintero O.A. Ellenberg J. Baum B. Myo19 ensures symmetric partitioning of mitochondria and coupling of mitochondrial segregation to cell division.Curr. Biol. 2014; 24: 2598-2605Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 13Shneyer B.I. Usaj M. Henn A. Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia.J. Cell Sci. 2016; 129: 543-556Crossref PubMed Scopus (46) Google Scholar, 14Hawthorne J.L. Mehta P.R. Singh P.P. Wong N.Q. Quintero O.A. Positively charged residues within the MYO19 MyMOMA domain are essential for proper localization of MYO19 to the mitochondrial outer membrane.Cytoskeleton (Hoboken). 2016; 73: 286-299Crossref PubMed Scopus (16) Google Scholar, 15Shneyer B.I. Usaj M. Wiesel-Motiuk N. Regev R. Henn A. ROS induced distribution of mitochondria to filopodia by Myo19 depends on a class specific tryptophan in the motor domain.Sci. Rep. 2017; 7: 11577Crossref PubMed Scopus (26) Google Scholar, 16Lopez-Domenech G. Covill-Cooke C. Ivankovic D. Halff E.F. Sheehan D.F. Norkett R. Birsa N. Kittler J.T. Miro proteins coordinate microtubule- and actin-dependent mitochondrial transport and distribution.EMBO J. 2018; 37: 321-336Crossref PubMed Scopus (174) Google Scholar, 17Oeding S.J. Majstrowicz K. Hu X.P. Schwarz V. Freitag A. Honnert U. Nikolaus P. Bahler M. Identification of Miro1 and Miro2 as mitochondrial receptors for myosin XIX.J. Cell Sci. 2018; 131: jcs219469Crossref PubMed Scopus (62) Google Scholar). The C-terminal tail domain of Myo19 is necessary and sufficient for mitochondria targeting (11Quintero O.A. DiVito M.M. Adikes R.C. Kortan M.B. Case L.B. Lier A.J. Panaretos N.S. Slater S.Q. Rengarajan M. Feliu M. Cheney R.E. Human Myo19 is a novel myosin that associates with mitochondria.Curr. Biol. 2009; 19: 2008-2013Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 13Shneyer B.I. Usaj M. Henn A. Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia.J. Cell Sci. 2016; 129: 543-556Crossref PubMed Scopus (46) Google Scholar, 14Hawthorne J.L. Mehta P.R. Singh P.P. Wong N.Q. Quintero O.A. Positively charged residues within the MYO19 MyMOMA domain are essential for proper localization of MYO19 to the mitochondrial outer membrane.Cytoskeleton (Hoboken). 2016; 73: 286-299Crossref PubMed Scopus (16) Google Scholar). It is demonstrated that the C-terminal tail domain binds to outer mitochondrial rho GTPase protein (Miro1 and/or Miro2) (17Oeding S.J. Majstrowicz K. Hu X.P. Schwarz V. Freitag A. Honnert U. Nikolaus P. Bahler M. Identification of Miro1 and Miro2 as mitochondrial receptors for myosin XIX.J. Cell Sci. 2018; 131: jcs219469Crossref PubMed Scopus (62) Google Scholar), thus protecting Myo19 from degradation (16Lopez-Domenech G. Covill-Cooke C. Ivankovic D. Halff E.F. Sheehan D.F. Norkett R. Birsa N. Kittler J.T. Miro proteins coordinate microtubule- and actin-dependent mitochondrial transport and distribution.EMBO J. 2018; 37: 321-336Crossref PubMed Scopus (174) Google Scholar). Miro proteins also associate with KIF5 (also called kinesin 1) and dynein–dynactin complex, microtubule-associated motor proteins (18Stowers R.S. Megeath L.J. Gorska-Andrzejak J. Meinertzhagen I.A. Schwarz T.L. Axonal transport of mitochondria to synapses depends on milton, a novel Drosophila protein.Neuron. 2002; 36: 1063-1077Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar, 19Glater E.E. Megeath L.J. Stowers R.S. Schwarz T.L. Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent.J. Cell Biol. 2006; 173: 545-557Crossref PubMed Scopus (497) Google Scholar, 20van Spronsen M. Mikhaylova M. Lipka J. Schlager M.A. van den Heuvel D.J. Kuijpers M. Wulf P.S. Keijzer N. Demmers J. Kapitein L.C. Jaarsma D. Gerritsen H.C. Akhmanova A. Hoogenraad C.C. TRAK/Milton motor-adaptor proteins steer mitochondrial trafficking to axons and dendrites.Neuron. 2013; 77: 485-502Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 21Gama J.B. Pereira C. Simoes P.A. Celestino R. Reis R.M. Barbosa D.J. Pires H.R. Carvalho C. Amorim J. Carvalho A.X. Cheerambathur D.K. Gassmann R. Molecular mechanism of dynein recruitment to kinetochores by the Rod-Zw10-Zwilch complex and Spindly.J. Cell Biol. 2017; 216: 943-960Crossref PubMed Scopus (80) Google Scholar). Therefore, it is anticipated that Miro proteins coordinate both microtubule-based mitochondria movement by KIF5/dynein and actin-based movement by Myo19 in cells. Recent studies have also revealed that Myo19 migrates to the tip of filopodia in cells by stimulations such as glucose starvation and reactive oxygen species (13Shneyer B.I. Usaj M. Henn A. Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia.J. Cell Sci. 2016; 129: 543-556Crossref PubMed Scopus (46) Google Scholar, 15Shneyer B.I. Usaj M. Wiesel-Motiuk N. Regev R. Henn A. ROS induced distribution of mitochondria to filopodia by Myo19 depends on a class specific tryptophan in the motor domain.Sci. Rep. 2017; 7: 11577Crossref PubMed Scopus (26) Google Scholar). This suggests that Myo19 may be regulated by such stimulations. Biochemical studies have revealed that Myo19 has actin-activated ATPase activity like most other myosins (22Adikes R.C. Unrath W.C. Yengo C.M. Quintero O.A. Biochemical and bioinformatic analysis of the myosin-XIX motor domain.Cytoskeleton (Hoboken). 2013; 70: 281-295Crossref PubMed Scopus (23) Google Scholar, 23Lu Z. Ma X.N. Zhang H.M. Ji H.H. Ding H. Zhang J. Luo D. Sun Y. Li X.D. Mouse myosin-19 is a plus-end-directed, high-duty ratio molecular motor.J. Biol. Chem. 2014; 289: 18535-18548Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 24Usaj M. Henn A. Kinetic adaptation of human Myo19 for active mitochondrial transport to growing filopodia tips.Sci. Rep. 2017; 7: 11596Crossref PubMed Scopus (15) Google Scholar) with a plus (barbed)-end-directed (23Lu Z. Ma X.N. Zhang H.M. Ji H.H. Ding H. Zhang J. Luo D. Sun Y. Li X.D. Mouse myosin-19 is a plus-end-directed, high-duty ratio molecular motor.J. Biol. Chem. 2014; 289: 18535-18548Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) high duty ratio motor (23Lu Z. Ma X.N. Zhang H.M. Ji H.H. Ding H. Zhang J. Luo D. Sun Y. Li X.D. Mouse myosin-19 is a plus-end-directed, high-duty ratio molecular motor.J. Biol. Chem. 2014; 289: 18535-18548Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 24Usaj M. Henn A. Kinetic adaptation of human Myo19 for active mitochondrial transport to growing filopodia tips.Sci. Rep. 2017; 7: 11596Crossref PubMed Scopus (15) Google Scholar). The IQ domain of Myo19 binds to CaM (a common light chain of unconventional myosins) as well as myosin regulatory light chains (23Lu Z. Ma X.N. Zhang H.M. Ji H.H. Ding H. Zhang J. Luo D. Sun Y. Li X.D. Mouse myosin-19 is a plus-end-directed, high-duty ratio molecular motor.J. Biol. Chem. 2014; 289: 18535-18548Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). A critical unanswered question is whether Myo19 is a motor protein that is suitable for cargo transportation. As Myo19 exclusively colocalizes with mitochondria, it is anticipated that Myo19 can transport mitochondria if Myo19 has a suitable motor characteristic for cargo transportation (15Shneyer B.I. Usaj M. Wiesel-Motiuk N. Regev R. Henn A. ROS induced distribution of mitochondria to filopodia by Myo19 depends on a class specific tryptophan in the motor domain.Sci. Rep. 2017; 7: 11577Crossref PubMed Scopus (26) Google Scholar, 24Usaj M. Henn A. Kinetic adaptation of human Myo19 for active mitochondrial transport to growing filopodia tips.Sci. Rep. 2017; 7: 11596Crossref PubMed Scopus (15) Google Scholar). Since Myo19 does not have a coiled coil, it has been thought that Myo19 alone is a single-headed motor (2Odronitz F. Kollmar M. Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species.Genome Biol. 2007; 8: R196Crossref PubMed Scopus (291) Google Scholar). It is puzzling whether a single-headed Myo19 can move on actin filaments with well-known hand-over-hand mechanism (9Yildiz A. Forkey J.N. McKinney S.A. Ha T. Goldman Y.E. Selvin P.R. Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization.Science. 2003; 300: 2061-2065Crossref PubMed Scopus (1586) Google Scholar). The critical missing information is whether Myo19 can move processively in a way that is suitable for transportation of mitochondria, since accumulation of Myo19 at filopodial tips does not necessarily assure the processive movement of Myo19. The aim of this study is to clarify whether human Myo19 (HM19) can move processively on actin filaments and transport mitochondria in cells. To clarify the movement of Myo19 and its role in mitochondrial movement, we utilized single-molecule analysis with a highly sensitive total internal reflection fluorescence (TIRF) microscope by highly inclined and laminated optical sheet (HILO) illumination (25Ambrose E.J. A surface contact microscope for the study of cell movements.Nature. 1956; 178: 1194Crossref PubMed Scopus (80) Google Scholar, 26Sako Y. Minoguchi S. Yanagida T. Single-molecule imaging of EGFR signalling on the surface of living cells.Nat. Cell Biol. 2000; 2: 168-172Crossref PubMed Scopus (768) Google Scholar, 27Tokunaga M. Imamoto N. Sakata-Sogawa K. Highly inclined thin illumination enables clear single-molecule imaging in cells.Nat. Methods. 2008; 5: 159-161Crossref PubMed Scopus (873) Google Scholar). We characterized the movement of Myo19 at a single-molecule level using demembraned and living cell systems. The movement of single Myo19 molecules was further determined in in vitro single-molecule motility assay using single actin filaments by TIRF microscopy. Based upon our findings, we propose that Myo19 is a weak gaiting processive motor, and monomer–dimer transition of Myo19 via its tail domain is critical not only for the movement of single Myo19 molecules on actin filaments but also for actin-based mitochondrial movement in cells. To test the single-molecule movement of Myo19, we made various HM19 constructs (Fig. 1). To visualize HM19 molecules in living cells at a single-molecule level, we employed HaloTag technique. Advantage of this technique is that we can control the number of fluorescently labeled molecules within the cells by manipulating the concentration of exogenously added HaloTag ligand. HaloTag was added to the N-terminal end of HM19 to avoid possible interference of the HaloTag moiety on the interaction of HM19 and its binding partners such as mitochondria or possible influence on interheavy chain dimer formation (Fig. 1). As shown in Figure 1, there is no obvious predicted coiled-coil domain in HM19. It has been shown that mammalian myosin 6, myosin 7a, and myosin 10 do not readily form a dimer, even though they contain a predicted coiled-coil domain (4Umeki N. Jung H.S. Sakai T. Sato O. Ikebe R. Ikebe M. Phospholipid-dependent regulation of the motor activity of myosin X.Nat. Struct. Mol. Biol. 2011; 18: 783-788Crossref PubMed Scopus (78) Google Scholar, 28Lister I. Schmitz S. Walker M. Trinick J. Buss F. Veigel C. Kendrick-Jones J. A monomeric myosin VI with a large working stroke.EMBO J. 2004; 23: 1729-1738Crossref PubMed Scopus (138) Google Scholar, 29Umeki N. Jung H.S. Watanabe S. Sakai T. Li X.D. Ikebe R. Craig R. Ikebe M. The tail binds to the head-neck domain, inhibiting ATPase activity of myosin VIIA.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 8483-8488Crossref PubMed Scopus (68) Google Scholar), and it is thought that dimer formation of these myosins is facilitated by the binding of target molecules (4Umeki N. Jung H.S. Sakai T. Sato O. Ikebe R. Ikebe M. Phospholipid-dependent regulation of the motor activity of myosin X.Nat. Struct. Mol. Biol. 2011; 18: 783-788Crossref PubMed Scopus (78) Google Scholar, 5Sakai T. Umeki N. Ikebe R. Ikebe M. Cargo binding activates myosin VIIA motor function in cells.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 7028-7033Crossref PubMed Scopus (54) Google Scholar, 30Spudich G. Chibalina M.V. Au J.S. Arden S.D. Buss F. Kendrick-Jones J. Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2.Nat. Cell Biol. 2007; 9: 176-183Crossref PubMed Scopus (169) Google Scholar, 31Phichith D. Travaglia M. Yang Z. Liu X. Zong A.B. Safer D. Sweeney H.L. Cargo binding induces dimerization of myosin VI.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 17320-17324Crossref PubMed Scopus (85) Google Scholar), and that dimer formation is critical for the continuous movement of these myosins (32Park H. Ramamurthy B. Travaglia M. Safer D. Chen L.Q. Franzini-Armstrong C. Selvin P.R. Sweeney H.L. Full-length myosin VI dimerizes and moves processively along actin filaments upon monomer clustering.Mol. Cell. 2006; 21: 331-336Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 33Sato O. Jung H.S. Komatsu S. Tsukasaki Y. Watanabe T.M. Homma K. Ikebe M. Activated full-length myosin-X moves processively on filopodia with large steps toward diverse two-dimensional directions.Sci. Rep. 2017; 7: 44237Crossref PubMed Scopus (9) Google Scholar, 34Sato O. Komatsu S. Sakai T. Tsukasaki Y. Tanaka R. Mizutani T. Watanabe T.M. Ikebe R. Ikebe M. Human myosin VIIa is a very slow processive motor protein on various cellular actin structures.J. Biol. Chem. 2017; 292: 10950-10960Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). To address the importance of possible dimer formation of HM19 on its movement and cargo transporter activity, we made the forced dimer constructs of HM19-leucine zipper (LZ) (Fig. 1). It has been reported that the tail domain of myosin can function as an intramolecular inhibitor domain (35Li X.D. Jung H.S. Mabuchi K. Craig R. Ikebe M. The globular tail domain of myosin Va functions as an inhibitor of the myosin Va motor.J. Biol. Chem. 2006; 281: 21789-21798Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 36Li X.D. Jung H.S. Wang Q. Ikebe R. Craig R. Ikebe M. The globular tail domain puts on the brake to stop the ATPase cycle of myosin Va.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 1140-1145Crossref PubMed Scopus (60) Google Scholar, 37Jung H.S. Komatsu S. Ikebe M. Craig R. Head-head and head-tail interaction: A general mechanism for switching off myosin II activity in cells.Mol. Biol. Cell. 2008; 19: 3234-3242Crossref PubMed Scopus (144) Google Scholar). Since the tail domain is involved in the regulation of dimer formation of various myosin family members (29Umeki N. Jung H.S. Watanabe S. Sakai T. Li X.D. Ikebe R. Craig R. Ikebe M. The tail binds to the head-neck domain, inhibiting ATPase activity of myosin VIIA.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 8483-8488Crossref PubMed Scopus (68) Google Scholar, 30Spudich G. Chibalina M.V. Au J.S. Arden S.D. Buss F. Kendrick-Jones J. Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2.Nat. Cell Biol. 2007; 9: 176-183Crossref PubMed Scopus (169) Google Scholar, 38Sakai T. Jung H.S. Sato O. Yamada M.D. You D.J. Ikebe R. Ikebe M. Structure and regulation of the movement of human myosin VIIA.J. Biol. Chem. 2015; 290: 17587-17598Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), it is plausible that the tail domain of Myo19 may play a role in dimer formation. In order to study the role of the tail domain in the Myo19 motor function, we also produced a tail-truncated mutant of HM19 construct (Fig. 1). Although the HaloTag technique assures a single fluorophore in a single HM19 heavy chain, fluorophore-unbound HM19 can be present. In addition to the HaloTag-HM19, therefore, we also produced enhanced GFP (EGFP)-HM19 constructs to observe the single-molecule movement of HM19 (Fig. 1). In order to study the single-molecule stepping of HM19 movement, C-terminal FLAG tag was introduced in aid of protein isolation, and a c-Myc tag was introduced to label a quantum dot (Qdot) at the C-terminal end of the molecule via anti-Myc antibodies conjugated with Qdot (Fig. 1). All constructs used in this study were in good agreement in terms of the molecular masses and the response to antibodies (Fig. S1). It has been reported that mitochondrial transport can be driven by kinesin–dynein motors (39Melkov A. Abdu U. Regulation of long-distance transport of mitochondria along microtubules.Cell. Mol. Life Sci. 2018; 75: 163-176Crossref PubMed Scopus (71) Google Scholar). On the other hand, it is unclear whether an actin-based motor, myosin such as HM19, can drive mitochondrial movement or transport. We first examined the mitochondrial movements on actin tracks to elucidate the physiological relevance of HM19 in mitochondrial movement in cells. To address this question, heavy mitochondrial fraction (HMF) was prepared from HM19-untransfected and HM19-transfected human embryonic kidney 293T (HEK293T) cells according to Shneyer et al. (13Shneyer B.I. Usaj M. Henn A. Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia.J. Cell Sci. 2016; 129: 543-556Crossref PubMed Scopus (46) Google Scholar), since HMF contains HM19 (13Shneyer B.I. Usaj M. Henn A. Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia.J. Cell Sci. 2016; 129: 543-556Crossref PubMed Scopus (46) Google Scholar). The isolated mitochondria vesicles were introduced into the demembraned U2OS cells, and the movement was observed. We first tested the localization of HMF in the absence of ATP to see whether HM19 is associated with cellular actin. After addition of MitoTracker-stained HMF obtained from untransfected HEK293T cells, the demembraned cells were observed (Fig. S2, A–C). The result clearly showed that the HMF colocalized with actin on demembraned cells, suggesting that mitochondrial membrane vesicles are associated with cellular actin tracks, presumably through HM19 that is known to be associated with mitochondria (11Quintero O.A. DiVito M.M. Adikes R.C. Kortan M.B. Case L.B. Lier A.J. Panaretos N.S. Slater S.Q. Rengarajan M. Feliu M. Cheney R.E. Human Myo19 is a novel myosin that associates with mitochondria.Curr. Biol. 2009; 19: 2008-2013Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 13Shneyer B.I. Usaj M. Henn A. Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia.J. Cell Sci. 2016; 129: 543-556Crossref PubMed Scopus (46) Google Scholar). We also prepared mitochondria vesicles from HEK293 cells expressing Halo-HM19-forced dimer (HM19FullLZ) and examined the association of Halo-HM19FullLZ with mitochondria and actin tracks. Halo-HM19FullLZ was mostly colocalized with mitochondria vesicles (Fig. S2, D–F). Moreover, Halo-HM19FullLZ showed notable colocalization with actin tracks (Fig. S2, G–I), suggesting the association of Halo-HM19FullLZ on mitochondria with actin tracks. Consistently, EGFP-HM19FullWT and EGFP-HM19FullLZ showed marked colocalization with mitochondria. Moreover, EGFP-HM19FullLZ showed notable localization at filopodia (Figs. S3 and S4). Next, we examined the movement of mitochondria on demembraned cells using HMF prepared from Halo-HM19FullLZ-overexpressed HEK293T cells (see the "Experimental procedures" section). Interestingly, HMF prepared from the Halo-HM19FullLZ-expressing cells moved on the cellular actin tracks in the presence of ATP (Fig. 2A and Movie S1). The mean run length and the velocity were 0.37 ± 0.09 μm, and 55.7 ± 5.9 nm/s (mean ± SEM, n = 48), respectively (Fig. 2, B, C and Table 1). The histogram of the velocity showed a wide distribution with the combination of two different peaks at ∼30 and ∼60 nm/s. On the other hand, we did not observe the movement of HMF obtained from HM19-untransfected cells in the presence of ATP. These results support that the HMF movement is driven by HM19.Table 1Run lengths and velocities of HM19 in demembraned U2OS cellsExamined samplesRun length (λ)Velocityμmnm s−1HMF-expressing Halo-HM19FullLZ0.37 ± 0.0927.8 ±1.0 and 61.9 ± 6.4(n = 48)(n = 48)Purified Halo-HM19FullLZ0.43 ± 0.0841.1± 6.4 and 73.3 ± 8.6(n = 55)(n = 55)Experiments were in the presence of 1 mM ATP (see the "Experimental procedures" section). Mean ± SEM was indicated. Open table in a new tab Experiments were in the presence of 1 mM ATP (see the "Experimental procedures" section). Mean ± SEM was indicated. We next asked whether HM19 can move at a single-molecule level on demembraned cells' actin tracks. For this experiment, we purified EGFP-HM19FullLZ from EGFP-HM19FullLZ-transfected HEK293T cells by anti-FLAG affinity chromatography, and the movement was observed with a TIRF microscope (see the "Experimental procedures" section). As shown in Figure 3A, the fluorescent spots of EGFP signals typically showed two-step photobleaching. This indicates that the fluorescent spots contain two EGFP molecules, which are presumably derived from a single dimer molecule of EGFP-HM19FullLZ containing an LZ module. The movement of EGFP-HM19FullLZ was monitored under the TIRF microscope in the presence of 1 mM ATP (Movie S2). The single-molecule EGFP-HM19FullLZ continuously moved on demembraned cellular actin tracks, and the typical time-lapse images of the movement are shown in Figure 3B. The histogram of the run length is shown in Figure 3C. The obtained result was best fit to a single exponential equation to yield an average run length of 0.43 ± 0.08 μm (SEM, n = 55; Table 1). The result indicates that EGFP-HM19FullLZ moved processively on
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