BicaudalD Actively Regulates Microtubule Motor Activity in Lipid Droplet Transport Kristoffer S. Larsen, Jing Xu, Silvia Cermelli, Zhanyong Shu, Steven P. Gross* Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America Abstract Background: A great deal of sub-cellular organelle positioning, and essentially all minus-ended organelle transport, depends on cytoplasmic dynein, but how dynein’s function is regulated is not well understood. BicD is established to play a critical role in mediating dynein function—loss of BicD results in improperly localized nuclei, mRNA particles, and a dispersed Golgi apparatus—however exactly what BicD’s role is remains unknown. Nonetheless, it is widely believed that BicD may act to tether dynein to cargos. Here we use a combination of biophysical and biochemical studies to investigate BicD’s role in lipid droplet transport during Drosophila embryogenesis. Methodology/Principal Findings: Functional loss of BicD impairs the embryo’s ability to control the net direction of droplet transport; the developmentally controlled reversal in transport is eliminated. We find that minimal BicD expression (near- BicDnull) decreases the average run length of both plus and minus end directed microtubule (MT) based transport. A point mutation affecting the BicD N-terminus has very similar effects on transport during cellularization (phase II), but in phase III (gastrulation) motion actually appears better than in the wild-type. Conclusions/Significance: In contrast to a simple static tethering model of BicD function, or a role only in initial dynein recruitment to the cargo, our data uncovers a new dynamic role for BicD in actively regulating transport. Lipid droplets move bi-directionally, and our investigations demonstrate that BicD plays a critical—and temporally changing—role in balancing the relative contributions of plus-end and minus-end motors to control the net direction of transport. Our results suggest that while BicD might contribute to recruitment of dynein to the cargo it is not absolutely required for such dynein localization, and it clearly contributes to regulation, helping activation/inactivation of the motors. Citation: Larsen KS, Xu J, Cermelli S, Shu Z, Gross SP (2008) BicaudalD Actively Regulates Microtubule Motor Activity in Lipid Droplet Transport. PLoS ONE 3(11): e3763. doi:10.1371/journal.pone.0003763 Editor: Francois Schweisguth, Institut Pasteur, France Received August 29, 2008; Accepted November 3, 2008; Published November 19, 2008 Copyright: 2008 Larsen et al. 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 by the NIH, grant GM64624 to SPG. Partial support was also provided by a Department of Health and Human Services, Public Health Services training grant GM-07311-29 (to K.S.L.). 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: sgross@uci.edu Introduction Dynein is involved in many cellular processes, including mitosis, nuclear migration, mRNA transport, mitochondrial transport, golgi positioning, endosomal motion, transport of a variety of axonal and dendritic vesicles, IF motion, and the transport of pathogens such as herpes viruses. In many cases, it appears to play dual roles: first, it is essential for transporting particular organelles to specific locations, and second, once the organelles are appropriately localized, dynein plays a role in anchoring them there. Many of these cargos are transported along microtubules in a bi-directional fashion, involving the activity of a plus-end motor such as a kinesin family member and the activity of the minus-end motion dynein. The regulation of this bi-directional transport, or even uni- directional dynein-based motion alone, is not well understood. However, past work has uncovered a role for the protein BicD in facilitating dynein function in a number of systems. In many of these systems, loss of BicD function results in mislocalization of the cargo. Most of these studies have not involved real-time imaging and analysis. Therefore, it has not been clear to what extent the loss of BicD function alters actual transport of the cargo in question, as opposed to what extent it plays an essential role in anchoring the cargo once it is appropriately delivered. BicD was originally identified in Drosophila, in a mutant screen for dominant maternal-effect proteins [1]. BicD mutant embryos exhibited severe developmental defects including the loss of positional information defining the anterior region of the embryo and thereby giving rise to bicaudal embryos. It was later established that this phenotype resulted primarily from the aberrant localization of oskar mRNA, a posterior determinant which inhibits the anterior factors bicoid and hunchback [2]. Further studies showed that this improper localization resulted from altered dynein-based transport of the mRNA particles [3]. BicD has been demonstrated to be a component of dynein based transport via genetic analysis in Drosophila [2,4,5]. In addition to its role in mRNA transport, BicD has been established to play a role in nuclear positioning in Drosophila [6] and in golgi positioning in mammalian tissue culture [7,8]. Molecular studies have provided some insight into BicD’s function. In mammals, two homologues of Bicaudal D, BICD1 and BICD2, are present [8,9]. BicD is highly conserved, albeit PLoS ONE | www.plosone.org 1 November 2008 | Volume 3 | Issue 11 | e3763