BicD Regulates Transport 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 repeats–without 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 BicD’s 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 BicD’s function within the context of bi- directional lipid droplet based transport. In contrast to other systems where BicD has been studied, the particular strength of the Figure 1. BicD structure and expression levels. A: Drosophila BicD is 782 amino acids in length. There are four heptad repeat motifs. The location and type of various BicD alleles used in this study are indicated. Starbursts represent point mutations, Xs represent premature stop codons, and horizontal bars represent specific domain deletions. B: A generalized map of the interacting regions of BicD. Dashed lines indicate an approximate position based on data from mammalian BicD homologs. Regions displayed represent published data and not necessarily minimal binding sites. C: Ovarian BicD levels in heat-shock rescued BicDnull ovaries are quite low compared to wild-type. Levels seen during repeat daily heat- shocks (t = 0), and three days post heat shock cessation (t = 3) are shown in comparison to wild type. Lanes were loaded with equivalent amounts of total lysate. D: Specifically examining ovarian BicD expression in t = 3 ovaries indicates expressions levels approximately 1% of that in wild-type. This is shown in comparison to a wild-type dilution series of lanes loaded with 1/32nd and 1/64th the amount of total protein loaded for the t = 3 lane. E: A comparison of embryonic BicD expression between heat shock rescued embryos whose mothers have been off heat shock for one versus three days. F: The decrease in ovarian levels results in a decrease in embryonic levels: a comparison of BicD levels in embryos from OrR females (left) and BicDnull/ t = 1 females (right). Lanes were loaded with equivalent amounts of total embryo lysate. Signal was detected with mixed anti-BicD (4C2, 1B11). G: Quantitation of overall reduction of BicD in t = 1 embryos relative to the wildtype. Both t = 1 lanes were loaded with equivalent amounts of total embryo lysate; OrR lanes for comparison were loaded with 1/32, 1/64, or 1/128th the relative amount of total sample loaded in the t = 1 lanes. The t = 1 lanes have approximately the same signal strength as the OrR lysate diluted 1/64, suggesting that in the t = 1 embryos there is approximately 1.6% of wildtype BicD levels. Signal is detected with mixed anti-BicD antibodies (4c2, 1B11). doi:10.1371/journal.pone.0003763.g001 PLoS ONE | www.plosone.org 2 November 2008 | Volume 3 | Issue 11 | e3763