BicD Regulates Transport Figure 6. An analysis of the BicDPA66 allele’s failure to rescue. A: Sequence analysis of the BicDPA66 allele confirmed, as expected, that there was a single point mutation resulting in a single divergence (A40V) from the wild-type amino acid sequence. Light blue regions represent exons. Introns are represented by the intervening thin black lines. Red bars represent the two fragments upon which sequence analysis was carried out. B– D: BicDPA66 expression levels are similar to wild-type (B). BicDPA66 also showed similar expression to other alleles that were capable of rescue such as BicD1 and BicDH2D (C, D). Equivalent amounts of total embryo lysate were loaded per lane for each independent comparison (B–D). E: In phase II, BicDPA66 analysis identified a total number of droplets per frame intermediate between the wild-type and the (‘t = 1’, ‘t = 3,4’) values. F: Also in phase II, the identified number of moving droplets in the BicDPA66 background was intermediate to the lower values and the higher wild-type values. G: In the embryo, both BicD+ and BicDPA66 appear to be phosphorylated as indicated by the resulting shift following phosphatase (C.I.P.) treatment. H–J: Particle tracking of BicDPA66 showed that phase II run lengths were identical to the BicDnull (t = 3) for both directions of transport (t- test, p.0.75 for both directions) while phase IIb run lengths were similar to wild-type (t-test, p.0.17) for both directions of transport (H, I). Phase III run lengths did not come back down from the phase IIb peak as seen in wild type. BicDPA66 displayed an inability to reverse the net direction of transport, while also displaying a slight bias for net plus-end directed transport (J). doi:10.1371/journal.pone.0003763.g006 use biochemistry (e.g. droplet purifications) on the BicDPA66 embryos to confirm that the BicDPA66 protein localizes to the droplets. Further, immunolocalization studies were inconclusive because BicD serves many roles, so we observe punctae both close- to and away from the droplets in both wild-type and BicDPA66 mutant embryos (not shown). However, from a functional point of view, tracking analysis indicates that droplet motion (and hence BicDPA66 function) in Phase IIb is similar to wild type (Fig. 6H, I). This suggests that, at least in phase IIb, BicD is still physically present on the droplet. Similarly, motion continues to be good in phase III BicDPA66 embryos. In phase III, BicDPA66 run lengths of both plus and minus direction are noticeably longer than wild type (Fig. 6H, I). Combined, these observations suggest that BicD’s ability to possibly initially recruit dynein to the cargo is separate from its latter ability to regulate or alter transport. Rather than suggesting that the A40V mutation of BicDPA66 simply alters its localization, the data is consistent with the hypothesis that this mutation leaves BicD’s initial ability to recruit Dynein partially intact, but alters BicD’s function by somehow changing its ability to later regulate motion. Such a hypothesis would be consistent with the point mutation being localized in a portion of the protein previously reported to interact with dynein [12]. Discussion Past studies have found numerous roles for BicD in contributing to distinct dynein-mediated processes, but the exact mechanism through which BicD contributes to appropriate minus-end transport is not yet well established. However, evidence in a number of systems suggests that BicD may be able to play a role in PLoS ONE | www.plosone.org 8 November 2008 | Volume 3 | Issue 11 | e3763