BicD Regulates Transport Figure 5. Examining BicD alleles for the ability to rescue the reclouding defect of a low BicD background. A–I: Alleles comprised of domain deletions, point mutations, and premature stop codons were examined. B–C: Negative and positive controls are shown for comparison. All alleles appeared to rescue (C–G), with the exception of the point mutation BicDPA66 (H–I). Rescuing with two copies of BicDPA66 displayed a continued failure to rescue (I). E: On average, there is the suggestion that there may be only a partial rescue phenotype seen in the BicDH3D rescue background. Images with purple asterisks are those with a BicD-null phenotype. doi:10.1371/journal.pone.0003763.g005 account for the failure of the BicDPA66 allele to rescue the altered droplet distribution. A second possibility is that BicD function is important for appropriate arrangement of the microtubule cytoskeleton, so that the altered droplet transport in the BicDPA66 embryos could reflect a generalized microtubule defect. However, this is unlikely here because BicDPA66 and BicDnull (t = 3) embryos have nearly identical tracking phenotypes in phase II (t-test, p.0.75 for both directions; Fig. 6H, I), and confocal images of phase II microtubules in BicDnull (t = 3) embryos generally appeared fine (Fig. 2E). Additionally, the short run lengths in BicDPA66 phase II embryos transition to long wild-type run lengths of phase IIb within ,4 minutes, which would imply a rapidly repaired microtubule array, which seems unlikely. Overall, it seems unlikely that the short run lengths in phase II of BicDPA66 and the t = 3 are attributable to microtubule defects. A third possibility is that the failure of the BicDPA66 allele to rescue motion reflects the impaired localization of BicDPA66 to the droplet, due to the PA66 point mutation. To investigate this possibility, we considered a number of lines of evidence. First, others’ structural studies have suggested that BicD typically attaches to cargos via its C-terminal domain, but our sequencing analysis (Fig. 6A) indicated that the location of the point mutation is indeed as expected, in BicD’s very N-terminal domain, far from the C-terminal cargo-attachment region. Further, the analysis found no additional mutations (e.g.in the C-terminal portion of the protein) that could account for altered localization. Second, early in development (before phase II, when we analyze motion), BicD may recruit some portion of the Dynein to the droplets. This hypothesis is consistent with past reports, and also with the observation that in phases II and IIb, the number of moving droplets in BicDnull (t = 3, 4) was decreased by approximately 106 as compared to wild-type (Fig. 6F). With this in mind, we looked at the number of droplets moving in the PA66 background, and while it was less than in wild-type, it was roughly 36 to 46 more than in the (t = 3, 4) background (Fig. 6F). Since the presence of BicD with the PA66 mutation resulted in significantly more moving droplets than for the BicDnull, it suggests that the PA66 form of the protein still has a reasonable ability to recruit dynein to the droplets, consistent with the hypothesis that BicDPA66 is indeed able to localize to the droplets (at least early in development). Unfortunately, because they are quite few in number, we cannot PLoS ONE | www.plosone.org 7 November 2008 | Volume 3 | Issue 11 | e3763