Dynein Transport in Cell Cycle Figure 3. Movement analysis of fluorescent foci in interphase cells. A. Time-dependent displacements of six spot-like foci in the cytosol. Centrifugal movement (toward cell periphery) was recorded as positive and centripetal movement (toward nucleus) as negative displacement. B. Examples of dynein fluorescent foci which reversed the direction during observation. C, D. Histograms of velocity distributions of the spot-like foci toward the centripetal (C) and centrifugal (D) directions. The continuous lines correspond to a Gaussian fitting with the best-fitting velocity indicated in each figure. E. Histogram of velocity distribution of the comet-like foci. F. Histogram of velocity distribution of EB1-GFP in interphase HeLa cells. doi:10.1371/journal.pone.0007827.g003 spindle MTs. In the end of cytokinesis, dynein fluorescence and moving foci were hardly seen in the spindle MTs (Fig. 5F). No dynein fluorescence was detected at the mid body. Behavior of cytoplasmic dynein visualized by fluorescent LIC1 The important issue using IC74-mfGFP as a functional probe for cytoplasmic dynein is whether the moving foci represent dynein complex containing the heavy chain. The fact that a part of the expressed IC74-mfGFP is not incorporated into dynein complex (see Fig. 1B) would raise a possibility that some or all of the moving foci might represent IC74 lacking dynein heavy chain. To check this possibility, we tried to visualize dynein by fluorescent LIC which is an essential component of dynein complex [1]. Recent study has demonstrated that IC74 and LIC bind to the heavy chain but not to each other [34]. Therefore, colocalization of IC74 and LIC would strongly support the presence of the heavy chain. We made expression constructs of human LIC1 which was tagged with mfGFP or mCherry at the C-terminus. The purified LIC1-mfGFP fraction from the HEK cell lysate contained three bands: a high molecular mass polypeptide (.250 kDa) probably of the heavy chain, LIC1-mfGFP (,90 kDa), and IC74 (,75 kDa) (Fig. 7A, right). Thus, LIC1-mfGFP is successfully incorporated into dynein complex, as true of IC74-mfGFP (Fig. 7A, left). The LIC1-mfGFP fraction did not contain 50–60 kDa polypeptides seen in the IC74-mfGFP fraction. This seems to be consistent with an idea that these polypeptides are endogenous LICs which can be displaced by LIC1-mfGFP. Localization and behavior of LIC1 was examined by transiently expressing LIC1-mCherry in IC74-mfGFP HeLa cells. In interphase cells, LIC1 exhibited good colocalization with IC74 in both the spot-like and the comet-like foci (Fig. 7B and Movie S9). Colocalization was also confirmed at the moving foci on mitotic spindle MTs (Fig. 7C). These findings strongly support that the moving foci both in interphase and during mitosis probably represent dynein complex containing the heavy chain. PLoS ONE | www.plosone.org 4 November 2009 | Volume 4 | Issue 11 | e7827