useful probe for functional dynein complex in living cells [20,21]. IC74 tightly binds heavy chains and provides binding sites for three light chains [22,23,24,25,26] and p150Glued, a subunit of dynactin [15,27]. Thus, IC74 is an important ‘scaffold’ protein for dynein complex formation. To know behavior of cytoplasmic dynein in living cells, we generated a stable HeLa cell line expressing green fluorescence protein (GFP)-tagged IC74. We demonstrate that GFP-tagged IC74 is incorporated into functional dynein complex and monitors a cell cycle-dependent dynamic behavior of cytoplasmic dynein. Our findings suggest that dynein is transported to the site of action in preparation for the following cellular events primarily by the MT-based transport. Results Characterization of HeLa cells stably expressing fluorescent IC74 We generated a HeLa cell line stably expressing fluorescent IC74 using a fusion protein of IC74 and multifunctional GFP (mfGFP). mfGFP is a GFP mutant in which multiple affinity tags are inserted in tandem into an internal loop, and thus a useful tool both for live- cell imaging and biochemical characterization of protein complex [28]. mfGFP was fused at the C-terminus of IC74, near the WD40 repeat that is involved in binding to the heavy chain (Fig. 1A). HeLa cells expressing IC74-mfGFP showed no apparent abnormality in morphology and growth (data not shown). Sucrose density gradient sedimentation of the HeLa cell lysate demonstrated that the expressed IC74-mfGFP was ,5% of endogenous IC74, and that about half of IC74-mfGFP was sedimented to heavy sucrose fractions, suggesting incorporation of IC74-mfGFP into dynein complex (Fig. 1B). Correspondingly, the purified IC74-mfGFP fraction by StrepTrap column chromatography contained a high molecular mass polypeptide (.250 kDa) probably of dynein heavy chain, in addition to IC74-mfGFP (,110 kDa) (Fig. 1C). Several polypeptides were also seen at 50–60 kDa range which could be LICs. The purified fraction also contained endogenous IC74, suggesting heterodimer formation of IC74-mfGFP with endogenous IC74. In in vitro MT-gliding assay, the purified IC74-mfGFP fraction exhibited a minus end-directed motor activity with an averaged velocity of 11146148 nm/sec (mean6SD, n = 80) (Fig. 1D). Taken together, these findings indicate that the expressed IC74-mfGFP is successfully incorporated into functional dynein complex. Behavior of cytoplasmic dynein in interphase cells Live-cell imaging of IC74-mfGFP HeLa cells was carried out by high-speed laser scanning confocal microscopy (see Materials and Methods). In interphase cell, IC74-mfGFP distributed as discrete foci which moved in the cytosol in addition to cytosolic nonstructural fluorescence (Fig. 2A and Movie S1). Labeling of MTs with mCherry-a-tubulin revealed that these foci moved along with MTs (Fig. 2B and Movie S2). We referred to the direction of moving foci as ‘centripetal’ and ‘centrifugal’ in which foci move toward the nucleus and the cell periphery, respectively. Based on their shape and behavior, we classified the discrete foci into two groups: ‘spot-like’ foci and ‘comet-like’ foci. The spot-like foci are round-shaped, seen throughout the cytosol, and exhibit rapid movement at both centripetal and centrifugal directions (Fig. 2C), whereas the comet-like foci are comet-shaped, seen mostly at the cell periphery, and slowly move centrifugally (Fig. 2D, see also Movie S1). The spot-like foci moved bi-directionally (Fig. 3A) and some foci reversed the direction during observation (Fig. 3B). Turning direction was frequently observed, probably due to crossing the Dynein Transport in Cell Cycle Figure 1. Incorporation of IC74-mfGFP into functional cyto- plasmic dynein complex. A. Schematic diagram of IC74-mfGFP. mfGFP was fused at the C-terminus of IC74. B. Sucrose density gradient sedimentation of lysate from IC74-mfGFP HeLa cells. Lysate was sedimented into 5–20% sucrose gradient. Fractions were analyzed by Western blotting with antibody specific to IC74 (74.1). IC74-mfGFP was ,5% of endogenous IC74. About half of IC74-mfGFP was sedimented to heavy sucrose fractions, indicating incorporation into dynein complex. C. SDS-PAGE and Western blotting of the purified IC74-mfGFP fraction. IC74-mfGFP was purified by StrepTrap column chromatography. The purified fraction contained a high molecular mass polypeptide (.250 kDa) probably of heavy chain (HC) in addition to IC74-mfGFP (,110 kDa). Polypeptides at 50–60 kDa range could be LICs. Endoge- nous IC74 was also detected, indicating heterodimer formation of IC74-mfGFP and IC74. IC74-mfGFP was detected by antibody specific to tetrahistidine which reacts with 86His tag in mfGFP. D. Histograms of velocity distribution of minus end-directed MTs in in vitro MT-gliding assay with the purified IC74-mfGFP fraction. doi:10.1371/journal.pone.0007827.g001 PLoS ONE | www.plosone.org 2 November 2009 | Volume 4 | Issue 11 | e7827