Dynein Transport in Cell Cycle Figure 7. Fluorescent LIC1 as a probe for cytoplasmic dynein. A. LIC1-mfGFP was purified from lysate of the LIC1-mfGFP expressing cells by StrepTrap chromatography and the protein composition was compared with that of the purified IC74-mfGFP fraction. Both heavy chain and IC74 were co-purified with LIC1-mfGFP. Polypeptides at 50–60 kDa range seen in the IC74-mfGFP fraction are undetectable in the purified LIC1-mfGFP fraction. B, C. IC74-mfGFP HeLa cells were transfected with LIC1-mCherry. Both the spot-like and the comet-like foci were labeled by LIC1-mCherry in interphase cells (B). Colocalization of IC74-mfGFP and LIC1-mCherry is also observed at mitotic spindle in metaphase cells (C). Scale bars: B, C, 10 mm. doi:10.1371/journal.pone.0007827.g007 (for interphase cells) or 1570 msec/image (for mitotic cells). The images were analyzed by Aquacosmos software (Hamamatsu Photonics, Japan). For movement analysis, the positions of the moving foci were determined by a two-dimensional Gaussian fitting algorithm with a custom program ‘Mark2’ [44]. The velocity of each foci was measured from displacements of .5 successive frames by a least-squares curve-fitting procedure. For paclitaxel treatment, cells were incubated for 10 min in KRH solution containing 10 mM paclitaxel. For methanol fixation, cells were washed twice with KRH solution, incubated for 15 min in ice- cold methanol, and rinsed with PBS before observations. Double thymidine treatment for synchronization of the HeLa cells at the G1/S boundary was carried out as described [45]. For multicolor observations, cells were observed using W-view system (Model 8509, Hamamatsu Photonics, Japan). Sucrose density gradient sedimentation Sucrose density gradient sedimentation of dynein complex was carried out as described [20] with some modifications. Briefly, lysate from IC74-mfGFP HeLa cells was prepared by homoge- nizing in a buffer A (0.1 M NaCl, 25 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.1 mM ATP, 0.5 mM dithiothreitol) containing 0.05% Triton X-100 and complete mini protease inhibitor cocktail (Roche). After centrifugation at 100,0006g for 30 min, the supernatant was applied onto top of a 5–20% sucrose gradient in buffer A. The gradient was ultracentrifuged for 24 h at 100,0006g. The resultant fractions of the gradient were processed to SDS-PAGE and Western blotting for detection of IC74. Purification of dynein complex with IC74-mfGFP Dynein complex was purified with SBP-tag in mfGFP [28]. Briefly, HeLa cells expressing IC74-mfGFP (ten 150 mm culture dishes) were collected and rinsed twice with phosphate buffered saline. The cell lysate was prepared by homogenizing the cells in buffer B (0.2 M NaCl, 50 mM Tris-HCl, pH 7.5, 10% sucrose, 5 mM MgCl2, 0.1 mM ATP, 0.5 mM dithiothreitol) containing 0.05% Triton X-100 and complete mini protease inhibitor cocktail (Roche). After centrifugation and filtration, the lysate was app- lied onto a StrepTrap HP column (GE healthcare) that had been equilibrated with buffer B. After washing with buffer A, bound proteins were eluted with buffer B containing 2.5 mM desthiobiotin. SDS-PAGE and Western blotting Proteins were separated by SDS-PAGE using standard Laemmli’s buffer system with 3–12% gradient gels and stained with Coomassie Brilliant Blue. For Western blotting, separated proteins were transferred onto PVDF membranes. Monoclonal antibodies against IC74 (74.1, Abcam, ab23905) and tetrahistidine (Qiagen, 34670) were used at 1:1,000 and 1:2,000 dilutions, respectively. Positive bands were detected by chemiluminescence using HRP-labeled anti-mouse IgG (KPL). In vitro MT gliding assay The MT gliding on the purified dynein-coated coverslips was observed under a dark-field microscope as described [44]. Supporting Information Movie S1 Cytoplasmic dynein in interphase. Many moving foci are observed. These foci are classified into two groups: spot-like foci and comet-like foci. Found at: doi:10.1371/journal.pone.0007827.s001 (3.35 MB MOV) Movie S2 Simultaneous observations of cytoplasmic dynein (green) and microtubules (red) in interphase. The discrete foci are moving along with the MTs. Found at: doi:10.1371/journal.pone.0007827.s002 (1.66 MB MOV) Movie S3 Behavior of dynein and EB1 at the cell periphery. Left, IC74-mfGFP; Right, EB1-GFP. Found at: doi:10.1371/journal.pone.0007827.s003 (1.79 MB MOV) PLoS ONE | www.plosone.org 8 November 2009 | Volume 4 | Issue 11 | e7827