Cell Cycle-Dependent Microtubule-Based Dynamic Transport of Cytoplasmic Dynein in Mammalian Cells Takuya Kobayashi, Takashi Murayama* Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan Abstract Background: Cytoplasmic dynein complex is a large multi-subunit microtubule (MT)-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. Methodology/Principal Findings: Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP)-tagged 74-kDa intermediate chain (IC74). IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs), suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. Conclusions and Significance: These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The 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. Citation: Kobayashi T, Murayama T (2009) Cell Cycle-Dependent Microtubule-Based Dynamic Transport of Cytoplasmic Dynein in Mammalian Cells. PLoS ONE 4(11): e7827. doi:10.1371/journal.pone.0007827 Editor: Anja-Katrin Bielinsky, University of Minnesota, United States of America Received June 29, 2009; Accepted October 15, 2009; Published November 13, 2009 Copyright: 2009 Kobayashi, Murayama. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants from the Japan Science and Technology Agency (SENTAN and X-ray free electron laser program) and Grants- in-Aid for Scientific Research, Japan Society for Promotion of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: takashim@juntendo.ac.jp Introduction Cytoplasmic dynein is a microtubule (MT) minus end-directed molecular motor and plays an important role in many cellular functions including organelle positioning, vesicle transport, and cell division [1]. Cytoplasmic dynein forms a huge multisubunit protein complex (,1.5 MDa) composed of two identical heavy chains, two 74-kDa intermediate chains (IC74), four light intermediate chains (LICs), and up to three light chain dimers (LC8, TcTex-1, and Roadblock) [2,3]. The heavy chain which is a member of the AAA ATPase protein superfamily carries a motor activity [4], and the other subunits comprising the cargo binding domain may play a regulatory role for intracellular transport [1]. In interphase cells, dynein transports vesicles, organelles, and other types of cargos from the cell periphery toward the cell center (to the minus end of MTs). This is opposite direction of transport by kinesin, a MT plus end-directed motor (from the cell center to the cell periphery). During mitosis, cytoplasmic dynein plays various roles, such as spindle organization, chromosome capture and congression, spindle assembly checkpoint (SAC) protein removal, and anaphase chromosome motility [5,6,7,8,9]. In accordance with these various roles, dynein is found at various sites, i.e., mitotic spindles, spindle poles, kinetochores, and cell cortex [10,11,12,13]. Kinetochore dynein may be important in chromosome capture and congression and SAC protein removal. The polar and cortical locations of dynein are consistent with roles in spindle assembly and positioning. Cytoplasmic dynein heavy chain is encoded by a single gene and is expressed stably and constitutively during the cell cycle [1]. This is in marked contrast to kinesins, which include a number of isoforms having different functions and are expressed in cell cycle- dependent manner [14]. To accomplish multiple functions, dynein interacts with various proteins which include dynactin [15], kinases [16], and endosomal proteins [17]. Dynactin is a large protein complex necessary for cargo binding of dynein and interact with various proteins, thus linking them with dynein [18,19]. These interactive partners might affect not only binding of the transport cargo, but also motor activity of dynein. Interaction with specific partners must be critically important in cell cycle- dependent roles of dynein. Although much information has accumulated about roles and regulations of cytoplasmic dynein as described above, it has not fully been understood about regulatory mechanism of cell cycle- dependent distribution of dynein in living cells. Direct visualization of dynein by live-cell imaging technique would greatly help understand this issue. It has recently been shown that IC74 is a PLoS ONE | www.plosone.org 1 November 2009 | Volume 4 | Issue 11 | e7827