BicaudalD Actively Regulates Microtubule Motor Activity in Lipid Droplet Transport

Citation: Larsen KS, Xu J, Cermelli S, Shu Z, Gross SP ( BicaudalD Actively Regulates Microtubule Motor Activity in Lipid Droplet Transport Kristoffer S. Larsen 0 Jing Xu 0 Silvia Cermelli 0 Zhanyong Shu 0 Steven P. Gross 0 Francois Schweisguth, Institut Pasteur, France 0 Department of Developmental and Cell Biology, University of California Irvine , Irvine, California , United States of America 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 (nearBicDnull) 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. - 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. 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 unidirectional 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 BicDs function. In mammals, two homologues of Bicaudal D, BICD1 and BICD2, are present [8,9]. BicD is highly conserved, albeit there is only one isoform in Drosophila. BicD is believed to exist in vivo as a homodimer [10,11]. Its C-terminus appears to bind to cargos [12], while also being known to foster specific dynein/ dynactin interactions [8,13] (Fig 1B). BicD is generally classified as having four well-defined coiled-coil regions (Fig. 1A) comprised of multiple heptad repeatswithout any other apparent motifs [11]. Overall, BicD has a flexible comma shaped structure with one large and one small globular domain and an apparently flexible linkage in between [10]. The large globular domain is believed to be at the C-terminus where binding to the cargo occurs. Mammalian BicD can directly interact with both Dynein and Dynactin, and because a cargo linked N-terminal fragment of BicD can tether dynein and subsequently induce dynein-based transport [12], it is believed that BicD may link dynein to the cargo. This is consistent with the studies of BicDs role in Drosophila mRNA transport, where overexpression of BicD leads to increased association of dynein with the mRNA particles [14]. Thus, one currently favored hypothesis is that BicD is a structural protein required for dynein localization. Tools have not existed to investigate the possibility that it might play important regulatory roles as well. Here, we investigate BicDs function within the context of bidirectional lipid droplet based transport. In contrast to other systems where BicD has been studied, the particular strength of the droplets is that it is possible to image them with high temporal and spatial resolution, allowing a careful study of how loss of BicD affects the properties of motion. Further, the possibility of biochemical purifications of droplets means that it is possible t (...truncated)