Dynein Transport in Cell Cycle Figure 6. Behavior of dynein in prometaphase and metaphase. A. Many fluorescent foci are moving on the spindle MTs in prometaphase. B. Methanol-fixed cell. These fluorescent foci are resistant to methanol fixation. C. Treatment with paclitaxel markedly reduced the moving foci, and visualized dynein at spindle poles and close to chromosomes (possibly kinetochores). D. Time-dependent displacements of the fluorescent foci on the spindles MTs. Movement from spindle poles toward chromosomes is recorded as positive displacement. E. Histogram of velocity distributions of the fluorescent foci on the spindle MTs. F. Simultaneous observations of IC74 and EB1 at the mitotic spindles in prometaphase. mCherry-EB1 was expressed in IC74-mfGFP HeLa cells. Fluorescent foci at the spindle MTs are colocalized with EB1. Some foci near chromosomes exhibit no colocalization with EB1, which might be kinetochore dynein. doi:10.1371/journal.pone.0007827.g006 primers: 59-GGGGAGCTCATGTCTGACAAAAGTGACTTA- AAA-39 and 59-GGGGGTACCGGCAGATAACTCAACAGT- 39, and subcloned into pIREShyg2 vector. mfGFP [28] was fused to the C terminus of IC74. cDNAs encoding a-tubulin, EB1, and LIC1 were amplified from HEK cells and subcloned into pcDNA5/FRT/TO vector. mCherry [43] was fused to either N-terminus (a-tubulin) or C terminus (EB1 and LIC1). Cell culture and transfection HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (Invitrogen) supplemented with 10% fetal calf serum and 2 mM L-glutamine. For generation of stable transfectants of IC74- mfGFP, the expression vector was transfected to HeLa cells using Lipofectamine LTX reagent (Invitrogen). The transfected cells were screened by hygromycin (400 mg/ml) for 2–3 weeks. Hygromycin- resistant colonies were further screened by fluorescence microscopy and GFP-positive colonies were selected. Transient expression of mCherry-tagged proteins was carried out using Lipofectamine LTX and assayed 24 h after transfection. Live cell imaging Live cell imaging was carried out with a high-speed confocal laser scanning microscope system. HeLa cells were grown on collagen-coated glass bottom dishes. Before observation, culture medium was changed to Krebs-Ringer Hepes (KRH) solution containing 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 2 mM NaHCO3, 0.5 mM MgSO4, 1 mM CaCl2, 10 mM glucose, and 10 mM Hepes, pH 7.4. The dish was placed on the stage of an inverted microscope with a Nipkow disc confocal laser scanning unit (CSU22, Yokogawa, Japan) equipped with an Argon Krypton Ion Laser (488 and 568 nm excitation). Cells were observed with a 1006 objective lens (Plan Apo, N.A. = 1.40, Nikon, Japan) at room temperature, and images were acquired with an EM-CCD camera (C9100, Hamamatsu Photonics, Japan) at a rate of 310 msec/image PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e7827