Figure 4. Levels of BicD on the lipid droplets decrease sharply with time. A: Lipids purified from staged one hour collections were probed for BicD and showed a large decrease in the amount of BicD on the droplet from phase I to III. Each lane was loaded with an equivalent amount of purified protein. B: During this same time, embryonic BicD levels are approximately constant. Each lane was loaded with an equivalent amount of total protein. C: Phase specific lipid purifications suggest that dynein levels on the droplets are approximately constant throughout phase I–III. Equivalent amounts of protein were loaded per lane. doi:10.1371/journal.pone.0003763.g004 suggesting that the amount of droplet-bound BicD is somehow directly regulated. Given the above t = 1/t = 3 results, and the observed changes in BicD levels, it seems likely that regulated changes in droplet-bound BicD levels could functionally contribute to the regulation of droplet transport and net directionality. Past studies reported that, when purposely attached to a cargo, an N-terminal fragment of mammalian BicD alone could recruit dynein to a given cargo [12]. Therefore, one appealing scenario is that droplet-bound BicD tethers dynein to the cargo, and thus droplet-bound BicD levels directly control transport by determining the amount of cargo- bound dynein. Then, regulating droplet-bound BicD levels could be used to regulate droplet-bound dynein levels, and potentially dynein activity. To investigate this possibility, we examined the amount of dynein bound to the droplets using staged lipid droplet purifications. DIC is hard to detect in western blots, so we have significantly more variability than for many other proteins. Nonetheless, we do not generally detect an obvious change in droplet-bound dynein levels as a function of developmental stage (Fig. 4C), suggesting there are likely not dramatic changes in the amount of dynein bound to the droplets in different developmental phases (though these measurements are not sensitive enough to detect subtle changes). Additionally, wild-type minus-end run lengths are approximately constant between phase I, II, and III [15], although they do increase briefly in the newly discovered phase IIb (Fig. 3C). Also, minus-end stalling forces (which reflect the number of engaged motors) have been shown to be higher in phase II than in phase I [15], although droplet-bound BicD is much lower in phase II than in phase I. Since the drastic fall in droplet BicD levels does not clearly coincide with altered minus- end run lengths in wild-type, or in reduced minus-end stalling forces, it seems unlikely that in phases I through III the majority of BicD would be playing an important role in tethering dynein to the cargos. To test the idea that BicD may not be absolutely essential for minus-end motility, we compared droplet motion in low (t = 1) and ,null (t = 3) BicD expression backgrounds. We observed minus- BicD Regulates Transport end motion that was statistically similar in the low and extremely low BicD backgrounds, despite the approximate six-fold difference in BicD expression (Fig. 1E). This suggests that by the time we reach t = 3 BicD levels, we are observing minus-end directed transport on droplets that are functionally for BicD, consistent with the idea that BicD may not be absolutely required for droplet motion. However, if BicD is important for initial recruitment of dynein, we might expect that the percentage of droplets moving to be decreased. Our automated analysis found that, in phase II, the number of moving droplets per frame was essentially the same between the t = 1 and t = 3 backgrounds, but quite reduced relative to the wild-type case (see below). These observations are consistent with a model that there is a basal amount of dynein that makes it to the droplets in a BicD-independent fashion, but that some of the droplet dynein initially is recruited via BicD (during embryogenesis), and that only droplets with dynein are able to transport themselves into the forming embryo. However, other interpretations are also possible (see discussion). Mutant analysis to gain insight into BicD’s functions Given the sudden BicD-dependent change in motion from phase II to IIB, it is possible that BicD has two roles—an initial structural one, possibly recruiting dynein as previously discussed by others, and a latter dynamic regulatory role, once the dynein is recruited. To further investigate this idea, and identify possibly separable functions of BicD, we undertook a structure-function study of a number of BicD mutants. We used a genetic strategy, wherein we evaluated whether specific mutations in BicD affected functions important for lipid droplet motion. Experimentally, our goal was to determine which alleles were capable of rescuing an expected reclouding defect (Fig. 5B) in embryos from female flies expressing very low levels of BicD. The rescue background, i.e. P{HA::BicD}/+; BicD2/BicD2, was expected to express ap- proximately 6% wild-type levels of BicD based on previous studies [11]. Our strategy involved making female flies, which in addition to the low levels of BicD, had BicD alleles that could potentially ‘‘rescue’’ the global reclouding defect. Using the female flies heterozygous for a HA-tagged BicD transgene (a gift from the lab of Dr. Ruth Steward), we examined several alleles comprised of small deletions and independent point mutations for their ability to rescue. Point mutants BicD1 (F684I) and BicD2 (E224K ) appeared to strongly rescue reclouding (Fig. 5F, G). A deletion of the second heptad repeat, from the N- terminus, rescued as well (Fig. 5D). A deletion of the third heptad repeat rescued, albeit perhaps not fully (Fig. 5E). By far, the clearest failure to rescue was provided by the A40V point mutant BicDPA66 (Fig. 5H, I). Thus, we decided to focus on a deeper understanding of the BicDPA66 allele. As part of this analysis, we tracked and analyzed droplets moving in BicDPA66 embryos in both phase II (N = 9 embryos, n = 122 droplets tracked), phase IIB (N = 5 embryos, n = 183 droplets tracked), and in phase III (N = 8 embryos, n = 236 droplets tracked). Further studies on the BicDPA66 allele Since BicD levels are important for overall function, one possibility was that the point mutation altered protein expression or stability. To test this possibility, we first used western blot analysis to determine BicD levels in the PA66 mutant embryos. We found that BicDPA66 expression levels were similar to wild-type (Fig. 6B). Further, rescuing with two copies of BicDPA66 instead of one displayed a continued reclouding defect (Fig. 5I). BicDPA66 also displays expression similar to the H2D and BicD1 alleles (Figs. 6C, D), both of which clearly rescue (Fig. 5D, F). Combined, these observations suggest that lowered BicD levels could not PLoS ONE | www.plosone.org 6 November 2008 | Volume 3 | Issue 11 | e3763