Figure 5. Behavior of cytoplasmic dynein in mitosis. A. Interphase. Dynein is distributed in the cytosol as the discrete foci. No fluorescence is detected in the centrosome and MTs. B. Late- interphase. Dynein is localized at the centrosome and radial MT array in addition to the moving foci. C. Prophase. Localization of dynein in the daughter centrosomes and radial MT arrays are remarkable. D. Metaphase. Spindle MTs are heavily stained by dynein fluorescence. Island-like foci are also observed near the cell cortex, which might be cortical dynein. E. Telophase. Dynein fluorescence increased in the cytosol with decrease on the spindle MTs. F. Cytokinesis. No apparent dynein fluorescence is detected on the spindle MTs nor mid body. Scale bars: A–F, 10 mm. doi:10.1371/journal.pone.0007827.g005 [35]. Interestingly, the comet-like foci of dynein were hardly seen near cell center where EB1 is present. Interaction of dynein and +TIPs is thought to be mediated by dynactin [35,40]. It is possible that dynein-dynactin and/or dynactin-+TIPs interactions may be temporally and spatially regulated. Visualization of dynactin or other +TIPs components would address the underlying mecha- nism of this regulation. Cytoplasmic dynein in late-interphase and prophase Cytoplasmic dynein is localized at centrosome in late-inter- phase. This localization is more profound in prophase. Cell cycle- dependent centrosome localization of dynein is consistent with the previous report by immunostaining of IC74 in COS-7 cells [37]. Dynactin is localized at centrosomes throughout the cell cycle [37]. Thus, centrosome localization of dynein might be regulated via interactions of dynein with dynactin and centrosomal components. In late-interphase and prophase, dynein fluorescence Dynein Transport in Cell Cycle was also observed on the radial MT array which seems to be relatively homogeneous and immotile. This might suggest direct or indirect interaction of dynein with MTs. Because such localization does not occur in interphase, some cycle-dependent changes might be involved in the interaction of dynein with MTs. Cytoplasmic dynein in prometaphase and metaphase We have demonstrated that cytoplasmic dynein is localized at three different sites in prometaphase and metaphase: on the spindle MTs, at kinetochores, and near cortex. These findings are well consistent with immunohistochemical localization of dynein with fixed cells: spindle dynein [10,11], kinetochore dynein [33,36], and cortical dynein [12]. Several novel findings have been obtained by live-cell imaging of these dyneins. On spindle MTs, majority of dynein is transported by +TIPs on the growing MTs. +TIPs-mediated transport of dynein also occurs on the astral MTs. Thus, +TIPs-mediated transport of dynein may be important for supply of dynein during mitosis. Kinetochore dynein is thought to be involved in attachment, orientation, and alignment of kinetochores to MTs [41]. We observed that kinetochore dynein is resistant to paclitaxel treatment and not colocalized with EB1, suggesting that kinetochore dynein may not be interacted with +TIPs. An interesting question is how kinetochore dynein is transported to the kinetochores. Cortical dynein is thought to be involved in proper spindle positioning during mitosis [42]. We found that several ‘island-like’ foci are localized near the cortical sites, which are linked with spindle poles by astral MTs, suggesting that they may be cortical dynein. Cortical dynein has been demonstrated by immunocytochemistry in MDCK cells, which is localized as spots at the cortex during late prometaphase and metaphase [12]. The island-like foci in our study, however, seem to be larger in size than the spots of cortical dynein observed with MDCK cells [12]. Difference in cell type could be one of possible reasons, because MDCK is a highly polarized cell line. Dynamic behavior of cytoplasmic dynein in cell cycle Based on the findings in the present study, we would like to propose that cytosolic dynein may be effectively transported to the site of action in preparation for the following cellular events. In interphase, dynein is transported to the cell periphery by +TIPs. This seems to be supply for the minus end-directed transport of various cargos. Dyneins at the centrosome and radial MT array in late-interphase and prophase may be prepared for formation of spindle poles and spindle MTs in prometaphase and metaphase. Dynein at the spindle poles, in turn, may be used for supply for the kinetochore and the cortical dyneins. It should be noted that dynein is transported primarily by the MT-based transport systems, i.e., +TIPs (as comet-like foci in interphase and moving foci on the spindle MTs) and MT binding (on the radial MT array in late- interphase). The distribution of dynein by MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein. It should also be noted that use of +TIPs and MT binding may be ‘cost effective’ compared with motor-driven transport in which no additional energies are required. Elucidation of the regulatory mechanisms of dynein transport will greatly help understand the MT-based intracellular transport system. Materials and Methods Generation of cDNA constructs cDNA encoding 74-kDa dynein intermediate chain (IC74) 1B isoform was amplified from HEK cells by RT-PCR using a pair of PLoS ONE | www.plosone.org 6 November 2009 | Volume 4 | Issue 11 | e7827