Dynein Transport in Cell Cycle Figure 4. Simultaneous observations of IC74 and EB1 in interphase cells. A. EB1-mCherry was expressed in IC74-mfGFP HeLa cells and behaviors of IC74 and EB1 were simultaneously observed using W-view system. B, C, D. Boxed areas in A are shown at higher magnification. Colocalization of the comet-like and EB1 are clearly observed at cell periphery (B, white box in A). The spot-like foci were not colocalized with EB1 (C, yellow box in A). The comet-like foci are hardly seen near cell center where EB1 is present (D, cyan box in A). Because focal plane was set close to bottom of the cell, microtubule networks under the nucleus are seen which cross the nucleus. Scale bars: A, 10 mm; B, C, 5 mm. doi:10.1371/journal.pone.0007827.g004 Discussion In this study, we have visualized cytoplasmic dynein with mfGFP-tagged IC74 in living HeLa cells. We found that behavior of cytoplasmic dynein dramatically changes in a cell-cycle dependent manner (Fig. 8). In interphase, dynein distributes as spot-like foci which might carry the cargos or as comet-like foci which are colocalized with EB1 on the growing MTs. In late- interphase, dynein is concentrated in the centrosome and the radial MT array. In prophase, localization at the daughter centrosomes and radial MT arrays is remarkable. In prometa- phase to metaphase, dynein is localized at spindle MTs where it frequently moves from spindle poles toward chromosomes and cortex. Possible kinetochore and cortical dyneins are also seen. In anaphase to telophase, spindle-associated dynein decreases, whereas cytosolic dynein increases. These findings correspond to the previous reports of localization of dynein by immunostaining of dynein subunits with fixed cells [10,11,12,33,35,36,37]. GFP-tagged IC74 as a functional probe for cytoplasmic dynein Use of IC74 as a functional probe for cytoplasmic dynein has initially been demonstrated with PC12 cells, in which all the stably expressed GFP-tagged IC74 is successfully incorporated into dynein complex [20]. A recent report, however, has claimed no incorporation of the transiently expressed GFP-tagged IC74 in dynein complex in COS-7 cells [38]. In the present study, we demonstrate that about half of the stably expressed IC74-mfGFP is incorporated into functional dynein complex. Because GFP tag is fused at the C-terminus of IC74 in all three studies, position of the GFP tag seems unlikely to be the reason for the difference. Cell type (PC12 and HeLa vs. COS-7) or expression system (stable expression vs. transient expression) could be possible cause. In our IC74-mfGFP HeLa cells, about half of IC74-mfGFP is not incorporated into dynein complex. One might assume that such unincorporated IC74-mfGFP affects the fluorescence measurements in live-cell imaging. Nevertheless, we believe that the moving foci in interphase (spot-like and comet-like foci) and those during mitosis (on the spindle MTs) represent dynein complex containing the heavy chain, because these foci were also labeled by LIC1, a component of dynein complex which is reported to bind to the heavy chain independently of IC74 [34]. Although cellular distribution of the unincorporated IC74-mfGFP remains unclear, it could partly be responsible for cytosolic nonstructural fluorescence. +TIPs-mediated transport of cytoplasmic dynein in interphase +TIPs are highly diverse group of the MT-associated proteins and thought to be involved in regulation of dynamic properties of MTs [31,32]. The MT plus-end accumulation of cytoplasmic dynein has been visualized in the fungus Aspergillus nidulans using GFP-tagged heavy chain nudA [39]. In mammalian cells, however, no live-cell imaging of dynein at the MT plus ends has so far been reported, although colocalization of endogenous IC74 with CLIP- 170 was shown in fixed COS-7 cells [35]. We here clearly demonstrated that cytoplasmic dynein complex is moving on MTs toward the cell periphery together with EB1. This supports the hypothesis of +TIPs-mediated transport of cytoplasmic dynein PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e7827