Movie S6 Relocalization of GFP-S332D/G697A myoB-S1DIQ in wild-type cells in response to local aspiration using a microcapillary. Speed: 506. This result is representative of 13 experiments. (AVI) Movie S7 Accumulation of GFP-myosin II along a cytoplasmic strand during cytokinesis C of a multinucleate pten- cell. The width of this field is 55 mm, and the speed is 356. (MOV) Text S1 Construction of the plasmids to express fluorescently labeled proteins. (DOC) References 1. Chhabra ES, Higgs HN (2007) The many faces of actin: matching assembly factors with cellular structures. Nat Cell Biol 9: 1110–1121. 2. Pollard TD, Cooper JA (2009) Actin, a central player in cell shape and movement. Science 326: 1208–1212. 3. McGough A (1998) F-actin-binding proteins. Curr Opin Struct Biol 8: 166–176. 4. Hild G, Bugyi B, Nyitrai M (2010) Conformational dynamics of actin: effectors and implications for biological function. Cytoskeleton (Hoboken) 67: 609–629. 5. Oda T, Maeda Y (2010) Multiple Conformations of F-actin. Structure 18: 761–767. 6. Galkin VE, Orlova A, Salmazo A, Djinovic-Carugo K, Egelman EH (2010) Opening of tandem calponin homology domains regulates their affinity for F- actin. Nat Struct Mol Biol 17: 614–616. 7. McGough A, Pope B, Chiu W, Weeds A (1997) Cofilin changes the twist of F- actin: implications for actin filament dynamics and cellular function. J Cell Biol 138: 771–781. 8. Galkin VE, Orlova A, Lukoyanova N, Wriggers W, Egelman EH (2001) Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits. J Cell Biol 153: 75–86. 9. Ressad F, Didry D, Xia GX, Hong Y, Chua NH, et al. (1998) Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G- and F- actins. Comparison of plant and human ADFs and effect of phosphorylation. J Biol Chem 273: 20894–20902. 10. Egelman EH, Francis N, DeRosier DJ (1982) F-actin is a helix with a random variable twist. Nature 298: 131–135. 11. Galkin VE, Orlova A, Schroder GF, Egelman EH (2010) Structural polymorphism in F-actin. Nat Struct Mol Biol 17: 1318–1323. 12. Fujii T, Iwane AH, Yanagida T, Namba K (2010) Direct visualization of secondary structures of F-actin by electron cryomicroscopy. Nature 467: 724–728. 13. Oosawa F, Fujime S, Ishiwata S, Mihashi K (1972) Dynamic properties of F- actin and thin filament. Cold Spring Harbor Symp Quant Biol 37: 277–285. 14. Thomas DD, Seidel JC, Gergely J (1979) Rotational dynamics of spin-labeled F- actin in the sub-millisecond time range. J Mol Biol 132: 257–273. 15. Tawada K (1969) Physicochemical studies of F-actin-heavy meromyosin solutions. Biochim Biophys Acta 172: 311–318. 16. Miki M, Wahl P, Auchet JC (1982) Fluorescence anisotropy of labeled F-actin: influence of divalent cations on the interaction between F-actin and myosin heads. Biochemistry 21: 3661–3665. 17. Loscalzo J, Reed GH, Weber A (1975) Conformational change and cooperativity in actin filaments free of tropomyosin. Proc Natl Acad Sci USA 72: 3412–3415. 18. Fujime S, Ishiwata S (1971) Dynamic study of F-actin by quasielastic scattering of laser light. J Mol Biol 62: 251–265. 19. Orlova A, Egelman EH (1997) Cooperative rigor binding of myosin to actin is a function of F-actin structure. J Mol Biol 265: 469–474. 20. Tokuraku K, Kurogi R, Toya R, Uyeda TQP (2008) Novel Mode of Cooperative Binding between Myosin and Mg2+-actin Filaments in the Presence of Low Concentrations of ATP. J Mol Biol 386: 149–162. 21. Sabry JH, Moores SL, Ryan S, Zang JH, Spudich JA (1997) Myosin heavy chain phosphorylation sites regulate myosin localization during cytokinesis in live cells. Mol Biol Cell 8: 2605–2615. 22. Egelhoff TT, Lee RJ, Spudich JA (1993) Dictyostelium myosin heavy chain phosphorylation sites regulate myosin filament assembly and localization in vivo. Cell 75: 363–371. 23. Uehara R, Goshima G, Mabuchi I, Vale RD, Spudich JA, et al. (2010) Determinants of myosin II cortical localization during cytokinesis. Curr Biol 20: 1080–1085. 24. Sussman M (1987) Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. In: Spudich JA, ed. Dictyostelium discoideum: Molecular approaches to Cell Biology. Orlando: Academic Press. pp 9–29. Stretching Actin Filaments Attracts Myosin II Text S2 filaments. (DOC) Acknowledgments Comparison of the fluorescent probes for actin We thank Drs. A. Ichihara and T. Hirano in the initial phase of the microscopy setup, Drs. K. Hirose, K. Tokuraku and T. Yasunaga for discussions and comments, and the National Bioresource Project Nenkin of Japan and Dicty Stock Center, USA for providing us with mutant Dictyostelium cell lines and cDNA clones. Author Contributions Conceived and designed the experiments: TU. Performed the experiments: TU YI NU AN SY. Analyzed the data: TU YI AN SY. Contributed reagents/materials/analysis tools: TU NU. Wrote the paper: TU. 25. Ruppel KM, Uyeda TQP, Spudich JA (1994) Role of highly conserved lysine 130 of myosin motor domain. J Biol Chem 269: 18773–18780. 26. Liu X, Ito K, Lee RJ, Uyeda TQP (2000) Involvement of tail domains in regulation of Dictyostelium myosin II. Biochem Biophys Res Commun 271: 75–81. 27. Yumura S, Mori S, Fukui Y (1984) Localization of actin and myosin for the study of amoeboid movement in Dictyostelium using improved immunofluores- cence. J Cell Biol 99: 894–899. 28. Pramanik MK, Iijima M, Iwadate Y, Yumura S (2009) PTEN is a mechanosensing signal transducer for myosin II localization in Dictyostelium cells. Genes Cells 14: 821–834. 29. Doyle T, Botstein D (1996) Movement of yeast cortical actin cytoskeleton visualized in vivo. Proc Natl Acad Sci USA 93: 3886–3891. 30. Westphal M, Jungbluth A, Heidecker M, Muhlbauer B, Heizer C, et al. (1997) Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein. Curr Biol 7: 176–183. 31. Asano Y, Mizuno T, Kon T, Nagasaki A, Sutoh K, et al. (2004) Keratocyte-like locomotion in amiB-null Dictyostelium cells. Cell Motil Cytoskeleton 59: 17–27. 32. Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, et al. (2008) Lifeact: a versatile marker to visualize F-actin. Nat Methods 5: 605–607. 33. Yumura S, Uyeda TQP (2003) Myosins and cell dynamics in cellular slime molds. Int Rev Cytol 224: 173–225. 34. Hibberd MG, Trentham DR (1986) Relationships between chemical and mechanical events during muscular contraction. Annu Rev Biophys Biophys Chem 15: 119–161. 35. Lymn RW (1979) Kinetic analysis of myosin and actomyosin ATPase. Annu Rev Biophys Bioeng 8: 145–163. 36. Batra R, Geeves MA, Manstein DJ (1999) Kinetic analysis of Dictyostelium discoideum myosin motor domains with glycine-to-alanine mutations in the reactive thiol region. Biochemistry 38: 6126–6134. 37. Patterson B, Ruppel KM, Wu Y, Spudich JA (1997) Cold-sensitive mutants G680V and G691C of Dictyostelium myosin II confer dramatically different biochemical defects. J Biol Chem 272: 27612–27617. 38. Kinose F, Wang SX, Kidambi US, Moncman CL, Winkelmann DA (1996) Glycine 699 is pivotal for the motor activity of skeletal muscle myosin. J Cell Biol 134: 895–909. 39. Uyeda TQP, Patterson B, Mendoza L, Hiratsuka Y (2002) Amino acids 519–524 of Dictyostelium myosin II form a surface loop that aids actin binding by facilitating a conformational change. J Muscle Res Cell Motil 23: 685–695. 40. Moores SL, Sabry JH, Spudich JA (1996) Myosin dynamics in live Dictyostelium cells. Proc Natl Acad Sci USA 93: 443–446. 41. De Lozanne A, Spudich JA (1987) Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science 236: 1086–1091. 42. Knecht DA, Loomis WF (1987) Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science 236: 1081–1086. 43. Uyeda TQP, Kitayama C, Yumura S (2000) Myosin II-independent cytokinesis in Dictyostelium: its mechanism and implications. Cell Struct Funct 25: 1–10. 44. Fukui Y, Lynch TJ, Brzeska H, Korn ED (1989) Myosin I is located at the leading edges of locomoting Dictyostelium amoebae. Nature 341: 328–331. 45. Bement WM, Mooseker MS (1995) TEDS rule: a molecular rationale for differential regulation of myosins by phosphorylation of the heavy chain head. Cell Motil Cytoskeleton 31: 87–92. 46. Effler JC, Iglesias PA, Robinson DN (2007) A mechanosensory system controls cell shape changes during mitosis. Cell Cycle 6: 30–35. 47. Merkel R, Simson R, Simson DA, Hohenadl M, Boulbitch A, et al. (2000) A micromechanic study of cell polarity and plasma membrane cell body coupling in Dictyostelium. Biophys J 79: 707–719. 48. Ren Y, Effler JC, Norstrom M, Luo T, Firtel RA, et al. (2009) Mechanosensing through Cooperative Interactions between Myosin II and the Actin Crosslinker Cortexillin I. Curr Biol 19: 1421–1428. PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e26200