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. 1995 Jul 2;130(2):345–354. doi: 10.1083/jcb.130.2.345

Actin-dependent mitochondrial motility in mitotic yeast and cell-free systems: identification of a motor activity on the mitochondrial surface

PMCID: PMC2199926  PMID: 7615636

Abstract

Using fluorescent membrane potential sensing dyes to stain budding yeast, mitochondria are resolved as tubular organelles aligned in radial arrays that converge at the bud neck. Time-lapse fluorescence microscopy reveals region-specific, directed mitochondrial movement during polarized yeast cell growth and mitotic cell division. Mitochondria in the central region of the mother cell move linearly towards the bud, traverse the bud neck, and progress towards the bud tip at an average velocity of 49 +/- 21 nm/sec. In contrast, mitochondria in the peripheral region of the mother cell and at the bud tip display significantly less movement. Yeast strains containing temperature sensitive lethal mutations in the actin gene show abnormal mitochondrial distribution. No mitochondrial movement is evident in these mutants after short-term shift to semi-permissive temperatures. Thus, the actin cytoskeleton is important for normal mitochondrial movement during inheritance. To determine the possible role of known myosin genes in yeast mitochondrial motility, we investigated mitochondrial inheritance in myo1, myo2, myo3 and myo4 single mutants and in a myo2, myo4 double mutant. Mitochondrial spatial arrangement and motility are not significantly affected by these mutations. We used a microfilament sliding assay to examine motor activity on isolated yeast mitochondria. Rhodamine-phalloidin labeled yeast actin filaments bind to immobilized yeast mitochondria, as well as unilamellar, right- side-out, sealed mitochondrial outer membrane vesicles. In the presence of low levels of ATP (0.1-100 microM), we observed F-actin sliding on immobilized yeast mitochondria. In the presence of high levels of ATP (500 microM-2 mM), bound filaments are released from mitochondria and mitochondrial outer membranes. The maximum velocity of mitochondria- driven microfilament sliding (23 +/- 11 nm/sec) is similar to that of mitochondrial movement in living cells. This motor activity requires hydrolysis of ATP, does not require cytosolic extracts, is sensitive to protease treatment, and displays an ATP concentration dependence similar to that of members of the myosin family of actin-based motors. This is the first demonstration of an actin-based motor activity in a defined organelle population.

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Selected References

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  1. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams R. J., Pollard T. D. Membrane-bound myosin-I provides new mechanisms in cell motility. Cell Motil Cytoskeleton. 1989;14(2):178–182. doi: 10.1002/cm.970140203. [DOI] [PubMed] [Google Scholar]
  3. Adams R. J., Pollard T. D. Propulsion of organelles isolated from Acanthamoeba along actin filaments by myosin-I. Nature. 1986 Aug 21;322(6081):754–756. doi: 10.1038/322754a0. [DOI] [PubMed] [Google Scholar]
  4. Brockerhoff S. E., Stevens R. C., Davis T. N. The unconventional myosin, Myo2p, is a calmodulin target at sites of cell growth in Saccharomyces cerevisiae. J Cell Biol. 1994 Feb;124(3):315–323. doi: 10.1083/jcb.124.3.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen L. B. Fluorescent labeling of mitochondria. Methods Cell Biol. 1989;29:103–123. doi: 10.1016/s0091-679x(08)60190-9. [DOI] [PubMed] [Google Scholar]
  6. Criddle R. S., Schatz G. Promitochondria of anaerobically grown yeast. I. Isolation and biochemical properties. Biochemistry. 1969 Jan;8(1):322–334. doi: 10.1021/bi00829a045. [DOI] [PubMed] [Google Scholar]
  7. Drubin D. G., Jones H. D., Wertman K. F. Actin structure and function: roles in mitochondrial organization and morphogenesis in budding yeast and identification of the phalloidin-binding site. Mol Biol Cell. 1993 Dec;4(12):1277–1294. doi: 10.1091/mbc.4.12.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fath K. R., Burgess D. R. Golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein. J Cell Biol. 1993 Jan;120(1):117–127. doi: 10.1083/jcb.120.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Glick B. S., Pon L. A. Isolation of highly purified mitochondria from Saccharomyces cerevisiae. Methods Enzymol. 1995;260:213–223. doi: 10.1016/0076-6879(95)60139-2. [DOI] [PubMed] [Google Scholar]
  10. Goodson H. V., Spudich J. A. Identification and molecular characterization of a yeast myosin I. Cell Motil Cytoskeleton. 1995;30(1):73–84. doi: 10.1002/cm.970300109. [DOI] [PubMed] [Google Scholar]
  11. Govindan B., Bowser R., Novick P. The role of Myo2, a yeast class V myosin, in vesicular transport. J Cell Biol. 1995 Mar;128(6):1055–1068. doi: 10.1083/jcb.128.6.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grolig F., Williamson R. E., Parke J., Miller C., Anderton B. H. Myosin and Ca2+-sensitive streaming in the alga Chara: detection of two polypeptides reacting with a monoclonal anti-myosin and their localization in the streaming endoplasm. Eur J Cell Biol. 1988 Oct;47(1):22–31. [PubMed] [Google Scholar]
  13. Haarer B. K., Petzold A., Lillie S. H., Brown S. S. Identification of MYO4, a second class V myosin gene in yeast. J Cell Sci. 1994 Apr;107(Pt 4):1055–1064. doi: 10.1242/jcs.107.4.1055. [DOI] [PubMed] [Google Scholar]
  14. Hegmann T. E., Schulte D. L., Lin J. L., Lin J. J. Inhibition of intracellular granule movement by microinjection of monoclonal antibodies against caldesmon. Cell Motil Cytoskeleton. 1991;20(2):109–120. doi: 10.1002/cm.970200204. [DOI] [PubMed] [Google Scholar]
  15. Huffaker T. C., Thomas J. H., Botstein D. Diverse effects of beta-tubulin mutations on microtubule formation and function. J Cell Biol. 1988 Jun;106(6):1997–2010. doi: 10.1083/jcb.106.6.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jacobs C. W., Adams A. E., Szaniszlo P. J., Pringle J. R. Functions of microtubules in the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1988 Oct;107(4):1409–1426. doi: 10.1083/jcb.107.4.1409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Johara M., Toyoshima Y. Y., Ishijima A., Kojima H., Yanagida T., Sutoh K. Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2127–2131. doi: 10.1073/pnas.90.6.2127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johnston G. C., Prendergast J. A., Singer R. A. The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles. J Cell Biol. 1991 May;113(3):539–551. doi: 10.1083/jcb.113.3.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kachar B. Direct visualization of organelle movement along actin filaments dissociated from characean algae. Science. 1985 Mar 15;227(4692):1355–1357. doi: 10.1126/science.4038817. [DOI] [PubMed] [Google Scholar]
  20. Kachar B., Reese T. S. The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin filaments. J Cell Biol. 1988 May;106(5):1545–1552. doi: 10.1083/jcb.106.5.1545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kilmartin J. V., Adams A. E. Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J Cell Biol. 1984 Mar;98(3):922–933. doi: 10.1083/jcb.98.3.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Koning A. J., Lum P. Y., Williams J. M., Wright R. DiOC6 staining reveals organelle structure and dynamics in living yeast cells. Cell Motil Cytoskeleton. 1993;25(2):111–128. doi: 10.1002/cm.970250202. [DOI] [PubMed] [Google Scholar]
  23. Kron S. J., Spudich J. A. Fluorescent actin filaments move on myosin fixed to a glass surface. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6272–6276. doi: 10.1073/pnas.83.17.6272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kuznetsov S. A., Langford G. M., Weiss D. G. Actin-dependent organelle movement in squid axoplasm. Nature. 1992 Apr 23;356(6371):722–725. doi: 10.1038/356722a0. [DOI] [PubMed] [Google Scholar]
  25. Kübler E., Riezman H. Actin and fimbrin are required for the internalization step of endocytosis in yeast. EMBO J. 1993 Jul;12(7):2855–2862. doi: 10.1002/j.1460-2075.1993.tb05947.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lazzarino D. A., Boldogh I., Smith M. G., Rosand J., Pon L. A. Yeast mitochondria contain ATP-sensitive, reversible actin-binding activity. Mol Biol Cell. 1994 Jul;5(7):807–818. doi: 10.1091/mbc.5.7.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lillie S. H., Brown S. S. Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae. J Cell Biol. 1994 May;125(4):825–842. doi: 10.1083/jcb.125.4.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mulholland J., Preuss D., Moon A., Wong A., Drubin D., Botstein D. Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane. J Cell Biol. 1994 Apr;125(2):381–391. doi: 10.1083/jcb.125.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Novick P., Botstein D. Phenotypic analysis of temperature-sensitive yeast actin mutants. Cell. 1985 Feb;40(2):405–416. doi: 10.1016/0092-8674(85)90154-0. [DOI] [PubMed] [Google Scholar]
  30. Palmer R. E., Sullivan D. S., Huffaker T., Koshland D. Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae. J Cell Biol. 1992 Nov;119(3):583–593. doi: 10.1083/jcb.119.3.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Prendergast J. A., Murray L. E., Rowley A., Carruthers D. R., Singer R. A., Johnston G. C. Size selection identifies new genes that regulate Saccharomyces cerevisiae cell proliferation. Genetics. 1990 Jan;124(1):81–90. doi: 10.1093/genetics/124.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rayment I., Holden H. M., Whittaker M., Yohn C. B., Lorenz M., Holmes K. C., Milligan R. A. Structure of the actin-myosin complex and its implications for muscle contraction. Science. 1993 Jul 2;261(5117):58–65. doi: 10.1126/science.8316858. [DOI] [PubMed] [Google Scholar]
  33. Read E. B., Okamura H. H., Drubin D. G. Actin- and tubulin-dependent functions during Saccharomyces cerevisiae mating projection formation. Mol Biol Cell. 1992 Apr;3(4):429–444. doi: 10.1091/mbc.3.4.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reuben J. P., Brandt P. W., Berman M., Grundfest H. Regulation of tension in the skinned crayfish muscle fiber. I. Contraction and relaxation in the absence of Ca (pCa is greater than 9). J Gen Physiol. 1971 Apr;57(4):385–407. doi: 10.1085/jgp.57.4.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Riezman H., Hay R., Gasser S., Daum G., Schneider G., Witte C., Schatz G. The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles. EMBO J. 1983;2(7):1105–1111. doi: 10.1002/j.1460-2075.1983.tb01553.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rodriguez J. R., Paterson B. M. Yeast myosin heavy chain mutant: maintenance of the cell type specific budding pattern and the normal deposition of chitin and cell wall components requires an intact myosin heavy chain gene. Cell Motil Cytoskeleton. 1990;17(4):301–308. doi: 10.1002/cm.970170405. [DOI] [PubMed] [Google Scholar]
  37. Schatz G., Saltzgaber J. Protein synthesis by yeast promitochondria in vivo. Biochem Biophys Res Commun. 1969 Dec 4;37(6):996–1001. doi: 10.1016/0006-291x(69)90230-7. [DOI] [PubMed] [Google Scholar]
  38. Schröder R. R., Manstein D. J., Jahn W., Holden H., Rayment I., Holmes K. C., Spudich J. A. Three-dimensional atomic model of F-actin decorated with Dictyostelium myosin S1. Nature. 1993 Jul 8;364(6433):171–174. doi: 10.1038/364171a0. [DOI] [PubMed] [Google Scholar]
  39. Sheetz M. P., Spudich J. A. Movement of myosin-coated fluorescent beads on actin cables in vitro. Nature. 1983 May 5;303(5912):31–35. doi: 10.1038/303031a0. [DOI] [PubMed] [Google Scholar]
  40. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  41. Shortle D., Novick P., Botstein D. Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4889–4893. doi: 10.1073/pnas.81.15.4889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Umemoto S., Sellers J. R. Characterization of in vitro motility assays using smooth muscle and cytoplasmic myosins. J Biol Chem. 1990 Sep 5;265(25):14864–14869. [PubMed] [Google Scholar]
  43. Watts F. Z., Shiels G., Orr E. The yeast MYO1 gene encoding a myosin-like protein required for cell division. EMBO J. 1987 Nov;6(11):3499–3505. doi: 10.1002/j.1460-2075.1987.tb02675.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weber A. Parallel response of myofibrillar contraction and relaxation to four different nucleoside triphophates. J Gen Physiol. 1969 Jun;53(6):781–791. doi: 10.1085/jgp.53.6.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wessels D., Schroeder N. A., Voss E., Hall A. L., Condeelis J., Soll D. R. cAMP-mediated inhibition of intracellular particle movement and actin reorganization in Dictyostelium. J Cell Biol. 1989 Dec;109(6 Pt 1):2841–2851. doi: 10.1083/jcb.109.6.2841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wessels D., Soll D. R. Myosin II heavy chain null mutant of Dictyostelium exhibits defective intracellular particle movement. J Cell Biol. 1990 Sep;111(3):1137–1148. doi: 10.1083/jcb.111.3.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Whitaker J. E., Moore P. L., Haugland R. P., Haugland R. P. Dihydrotetramethylrosamine: a long wavelength, fluorogenic peroxidase substrate evaluated in vitro and in a model phagocyte. Biochem Biophys Res Commun. 1991 Mar 15;175(2):387–393. doi: 10.1016/0006-291x(91)91576-x. [DOI] [PubMed] [Google Scholar]
  48. Zallen D. T., Burian R. M. On the beginnings of somatic cell hybridization: Boris Ephrussi and chromosome transplantation. Genetics. 1992 Sep;132(1):1–8. doi: 10.1093/genetics/132.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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