Figure 8. Schematic drawing of cell cycle-dependent dynamic behavior of cytoplasmic dynein in HeLa cells. In interphase, dynein may be associated with cargos which move bi-directionally (as the spot- like foci). A substantial population of dynein is transported toward cell periphery by +TIPs (as the comet-like foci). Dynein is not associated with the centrosome and MTs. In late-interphase, dynein is localized at the centrosome and the radial MT array. Centripetal movement of possible cargo-associated dynein is increased. In prophase, association of dynein with the daughter centrosomes and radial MT arrays are remarkable. In metaphase, dynein is associated with spindle poles and spindle MTs and is frequently transported toward chromosomes, probably by +TIPs. The motor-driven transport may also be present. In addition, dynein may be localized near chromosomes as kinetochore dynein and near cortex as cortical dynein. In telophase, spindle-associated dynein decreases, whereas cytosolic dynein increases. doi:10.1371/journal.pone.0007827.g008 Movie S4 Simultaneous observations of IC74-mfGFP (left, green in right) and EB1-mCherry (middle, red in right). The comet-like References 1. Vallee RB, Williams JC, Varma D, Barnhart LE (2004) Dynein: An ancient motor protein involved in multiple modes of transport. J Neurobiol 58: 189–200. 2. Pfister KK, Shah PR, Hummerich H, Russ A, Cotton J, et al. (2006) Genetic analysis of the cytoplasmic dynein subunit families. PLoS Genet 2: e1. 3. Pfister KK, Fisher EM, Gibbons IR, Hays TS, Holzbaur EL, et al. (2005) Cytoplasmic dynein nomenclature. J Cell Biol 171: 411–413. 4. Karata K, Inagawa T, Wilkinson A, Tatsuta T, Ogura T (1999) Dissecting the role of a conserved motif (the second region of homology) in the AAA family of ATPases: Site-directed mutagenesis of the ATP-dependent protease FtsH. J Biol Chem 274: 26225–26232. 5. Howell BJ, McEwen BF, Canman JC, Hoffman DB, Farrar EM, et al. (2001) Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation. J Cell Biol 155: 1159–1172. 6. Merdes A, Heald R, Samejima K, Earnshaw WC, Cleveland DW (2000) Formation of spindle poles by dynein/dynactin-dependent transport of NuMA. J Cell Biol 149: 851–862. Dynein Transport in Cell Cycle foci are colocalized wiht EB1, whereas the spot-like foci are not. The comet-like foci are hardly seen near the cell center, where EB1 is clearly observed. Found at: doi:10.1371/journal.pone.0007827.s004 (5.19 MB MOV) Movie S5 Centrosome and radial MT array localization of cytoplasmic dynein in late interphase cells. Found at: doi:10.1371/journal.pone.0007827.s005 (3.41 MB MOV) Movie S6 Cytoplasmic dynein in prometaphase. Many fluores- cent foci are moving from the spindle poles toward chromosomes. Possible astral MTs are also seen on which dynein is moving toward to the cortex. Island-like foci close to the cell cortex might be cortical dyneins. Note that fluorescent foci near the center which move back and forth might be kinetochore dynein. Found at: doi:10.1371/journal.pone.0007827.s006 (0.74 MB MOV) Movie S7 Cytoplasmic dynein in metaphase after treatment with paclitaxel. Paclitaxel (10 mM) caused a dramatic reduction in the spindle-associated dynein, and visualized dyneins at the spindle poles and kinetochores. Found at: doi:10.1371/journal.pone.0007827.s007 (1.10 MB MOV) Movie S8 Simultaneous observations of IC74-mfGFP (green) and EB1-mCherry (red) at mitotic spindles in prometaphase. Moving foci on the spindle MTs are colocalized with EB1, whereas foci staying near chromosomes (possible kinetochore dynein) is not. Found at: doi:10.1371/journal.pone.0007827.s008 (0.37 MB MOV) Movie S9 Simultaneous observations of IC74-mfGFP (left, green in right) and LIC1-mCherry (middle, red in right) in interphase cells. Both the spot-like and comet-like foci are colocalized with LIC1. Found at: doi:10.1371/journal.pone.0007827.s009 (5.47 MB MOV) Acknowledgments We thank Ken’ya Furuta (University of Tokyo) for kind help in MT gliding assay and generous gift of Mark2, and Roger Y. Tsien (Howard Hughes Medical Institute, UCSD) for providing a cDNA for mCherry. Author Contributions Conceived and designed the experiments: TK TM. Performed the experiments: TK TM. Analyzed the data: TK. Wrote the paper: TK TM. 7. Sharp DJ, Rogers GC, Scholey JM (2000) Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos. Nat Cell Biol 2: 922–930. 8. Bloom K (2001) Nuclear migration: cortical anchors for cytoplasmic dynein. Curr Biol 11: R326–329. 9. Banks JD, Heald R (2001) Chromosome movement: dynein-out at the kinetochore. Curr Biol 11: R128–131. 10. Pfarr C, Coue M, Grissom P, Hays TS, Porter ME, et al. (1990) Cytoplasmic dynein is localized to kinetochores during mitosis. Nature 345: 263–265. 11. Steuer ER, Wordeman L, Schroer TA, Sheetz MP (1990) Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature 345: 266–268. 12. Busson S, Dujardin D, Moreau A, Dompierre J, De Mey JR (1998) Dynein and dynactin are localized to astral microtubules and at cortical sites in mitotic epithelial cells. Curr Biol 8: 541–544. 13. O’Connell C, Wang Y (2000) Mammalian spindle orientation and position respond to changes in cell shape in a dynein-dependent fashion. Mol Biol Cell 11: 1765–1774. PLoS ONE | www.plosone.org 9 November 2009 | Volume 4 | Issue 11 | e7827