Figure 2. Live cell imaging of cytoplasmic dynein in IC74-mfGFP HeLa cells. A. Dynein is observed as moving fluorescent foci in the cytosol (see Movie S1). B. IC74-mfGFP HeLa cells were transfected with the mCherry-a-tubulin expression vector. Green and red represent dynein and MTs, respectively. Fluorescent foci are moving along with MTs (see Movie S2). C, D. Boxed areas in A are shown at higher magnification representing two classes of moving foci: spot-like foci (C, yellow box in A) and comet-like foci (D, white box in A). E. Distribution of dynein foci in methanol-fixed cells. The comet-like foci became clearly visible by disappearance of the spot-like foci. Scale bars: A, B, D, 10 mm; D, E, 5 mm. doi:10.1371/journal.pone.0007827.g002 intersecting MTs. In addition, some foci exhibited jiggling motion with no net motility (Movie S1). The velocity measurement by frame-to-frame tracking of the sequential images revealed that velocities of the spot-like foci at centripetal and centrifugal direction were 6916233 nm/sec (n = 43) and 6766214 nm/sec (n = 65), respectively (Figs. 3C and 3D). Some foci changed their velocity during movement (green squares in Fig. 3A). The properties of the spot-like foci are similar to those determined by fluorescent ligands for plasma membrane receptors that represent some endosomes [29,30]. The spot-like foci might be a population of dynein which carries cargos such as transport vesicles. The comet-like foci became clearly visible by methanol fixation of cells, which removes the spot-like foci (Fig. 2E). It completely disappeared by treatment of 10 mM paclitaxel that stabilizes MTs (data not shown). The velocity of the comet-like foci was 237652 nm/sec (n = 48), which was about one third of the velocity of the spot-like foci (Fig. 3E). These properties are very similar to those of the MT plus end-tracking proteins (+TIPs) [31,32]. Expression of the GFP-tagged EB1 in HeLa cells demonstrated similar behavior and velocity (235658 nm/sec, n = 51) to the comet-like foci (Figs. 3F and Movie S3). To further investigate relationships between dynein and EB1, we observed these proteins simultaneously by multicolor imaging. Expression of mCherry-tagged EB1 in IC74-mfGFP HeLa cells revealed that the comet-like foci were colocalized with EB1 near the cell periphery (Figs. 4A and 4B, and Movie S4). In contrast, Dynein Transport in Cell Cycle no colocalization was seen between the spot-like foci and EB1 (Fig. 4C). Interestingly, colocalization of IC74 and EB1 was clearly observed at the cell periphery but not near the cell center where the comet-like foci was hardly seen (Fig. 4D). Taken together, these findings suggest that cytoplasmic dynein may be transported to the cell periphery by interacting with +TIPs in interphase cells. Behavior of cytoplasmic dynein during mitosis We next determined behavior changes of cytoplasmic dynein during mitosis. IC74-mfGFP HeLa cells were synchronized at the G1/S boundary using double thymidine treatment. In late- interphase cells, the centrosome and the radial MT array became clearly visible by dynein fluorescence (Fig. 5B and Movie S5). This is in marked contrast to interphase cells in which the centrosome and the radial MT array were undetectable (Fig. 5A). This difference is unlikely to be due to changes in cell shape and centrosome position, because the centrosome and the radial MT array can be clearly seen by tubulin staining (see Fig. 2B). Dynein fluorescence on the radial MT array appeared to be relatively homogeneous and immotile. In addition, the spot-like foci moved on the radial MT array (Movie S5). Thus, dynein fluorescence on the radial MT array might be different from the moving foci. In late-interphase cells, centripetal movement of the spot-like foci appeared to be more frequent than in interphase. In prophase when the daughter centrosomes start to separate, dynein fluorescence on the daughter centrosomes and the radial MT array became more remarkable (Fig. 5C). The spot-like foci moved along with MTs as in interphase and late-interphase, whereas the comet-like foci were rarely detectable in prophase. In prometaphase and metaphase cells, dynein was localized at spindle MTs and spindle poles (Figs. 5D and 6A, and Movie S6). This fluorescence was primarily due to a number of fluorescent foci moving from spindle poles toward chromosomes. The velocity of moving foci was 97619 nm/sec 2 (n = 11) (Figs. 6D and 6E). The fluorescence remained unchanged after fixation with methanol (Fig. 6B). Treatment of cells with 10 mM paclitaxel caused a dramatic reduction in the number of moving foci (Fig. 6C and Movie S7). Correspondingly, the moving foci were colocalized with EB1 (Fig. 6F and Movie S8). These findings suggest that the moving foci on the spindle MTs may represent +TIPs-mediated transport of dynein. We also observed that some foci are moving on the spindle MTs at both directions even in the presence of paclitaxel (Movie S7). This suggests the presence of some motor-driven transports by kinesins or dynein on the spindle MTs. In addition to the moving foci, we observed some foci staying close to the chromosomes which are most prominent in prometaphase (Movies S6 and S8). These foci were still visible after paclitaxel treatment (Fig. 6C and Movie S7) and not colocalized with EB1 (Fig. 6F and Movie S8), suggesting no interactions with +TIPs. They might be kinetochore dynein [10,33]. Dynein was also localized as several ‘island-like’ foci near the cortical sites (Figs. 5D and 6A). Live-cell imaging at higher magnification revealed that these island-like foci are clusters of small distinct foci which seem to be weakly connected to each other (Movie S6). They were localized primarily parallel to the equatorial plane in prometaphase (Fig. 6A) and more widely distributed along with cortex in metaphase (Fig. 5D). These properties seem to be consistent with those of cortical dynein [12]. We found that a number of foci were moving from the spindle poles toward the cortical sites (Movie S6), suggesting possible transport of dynein along with the astral MTs to the cortex. In anaphase to telophase, dynein fluorescence increased in the cytosol (Fig. 5E). The moving foci were rarely observed on the PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e7827