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 to investigate additional aspects of BicD’s function. Importantly, while the droplets always move bi-directionally, they undergo developmentally controlled changes in net transport[15]. Early in development (nuclear division cycles 11–13) the droplets move but with no net transport (‘‘phase I’’). Then, in early cycle 14, the droplets move basally (net plus-end transport, ‘‘phase II’’). Finally, in later cycle 14 there is net minus-end transport (‘‘phase III’’). Thus, by studying changes in droplet-bound proteins at different times during development, it is in principle possible to gain insight into how transport is regulated[16]. We show that while BicD may help to recruit dynein to the cargo, it is not absolutely essential for at least partial dynein recruitment. Also, its role is more complicated than performing this task alone: during development, the majority of BicD leaves the droplet, but the dynein stays, and this subsequent departure of BicD does not obviously alter dynein function. Our measurements uncover a new role for BicD, in directly or indirectly contributing to plus-end motor activity. Finally, analysis of an intriguing BicD point mutation uncovers a clear ongoing temporally changing regulatory role for BicD. Results Because BicD is reported to play a role in either the transport or positioning of multiple organelles, and we have already established that our droplets are moved by dynein [17], we were interested in determining if BicD also played a role in lipid droplet transport. Globally, the distribution of lipid droplets in the embryo can be visualized using the neutral-lipid specific dye, Nile Red (Fig. 2C), and also by transmitted light microscopy (Fig. 2A, B). To investigate whether BicD played a role in droplet motion, we first wanted to decrease the overall amount of embryonic BicD, and determine whether this had any effect on droplet transport. Because the proteins in early embryos are predominantly maternally derived, and the complete loss of BicD is lethal, it has not been possible to generate embryos entirely lacking in BicD. Instead, we used a strategy based on a previously developed heat shock rescue technique to deliver just enough wild-type BicD to rescue otherwise BicDnull females [5]. Upon cessation of heat- shocking, ovarian BicD levels fell quickly. Continued for one hour per day, heat shocks of otherwise mothers produced significantly less ovarian BicD than did wild-type mothers (Fig. 1C). Also, by three to four days post heat shock (t = 3,4), ovarian BicD levels appeared to be slightly less than 1/64th wild type levels–i.e., approximately 1% of wild type (Fig. 1D). This loss of ovarian BicD resulted in a decrease in embryonic BicD (Fig. 1F); BicD in t = 1 embryos was approximately 1.6% that in wildtype embros (Fig. 1G). Additionally, in the low BicD- embryos, BicD levels continued to decrease as a function of time from heat shock: there is an approximate 6-fold difference between BicD levels at one day post heat shock (t = 1) and three to four days post heat shock (t = 3,4) (Fig. 1E). Thus, in t = 3,4 embryos, we calculate that BicD levels are ,0.267% of wild-type, i.e. have been reduced more than 3006. Using this technique therefore allowed us to achieve very low– and tunable–embryonic BicD levels. Examining the droplets in these embryos, we discovered that the loss of BicD had a number of effects. First, even when droplets are developmentally ‘clearing’ from the embryo’s edge (in Phase II), there were many fewer droplets obvious in this peripheral region of the low BicD embryos BicD Regulates Transport (Fig. 2K, L) as compared to wild-type (Fig. 2J, L). This could simply reflect a redistribution of droplets to the embryo center (where we are unable to quantify droplet distribution), or could reflects an overall decrease of embryonic droplets present in the embryo in this background. Second, loss of BicD severely altered the global droplet distribution (Fig. 2A–C): droplets cleared in phase II, but failed to re-cloud in phase III. To further understand how this altered global distribution of droplets came about, we considered a number of possibilities. The first was that loss of BicD led to an altered microtubule network in the embryos. To test this idea, we used confocal microscopy to visualize the microtubule network in fixed intact embryos. While the microtubules in the mutants appear slightly more disorganized, variation between different embryos is seen in the microtubule organization in both wild-type and mutants, and we observed no gross defects in the microtubules (Fig. 2E). To further test the viability of the microtubule network, we examined the distribution of another cargo, yolk vesicles, whose distribution was previously shown[18] to depend on intact microtubules. Their distribution was approximately normal (Fig. 2D). Thus, we hypothesize that the altered lipid droplet distribution was due to either altered droplet transport, or impaired ability to anchor the droplets once they were delivered. BicD likely contributes to appropriate droplet transport directly, through a role on the droplets Past studies have shown that the N-terminal portion of mammalian BicD can co-precipitate components of both the dynein and dynactin complexes [8,12]. Also, BicD homologs have been shown to directly bind dynein intermediate chain [13], the dynamitin subunit of dynactin [8], and kinesin [19] (see also Fig. 1B). This suggests that, for lipid droplets as well, BicD might facilitate appropriate transport via direct interaction with the transport machinery. To test whether BicD might play a direct role in droplet transport, we first investigated whether BicD was indeed present on the droplets. A first analysis involved purifying droplets, by spinning lipids from an embryo lysate through a simple sucrose step gradient. We saw, via western, that BicD signal was found on the lipids spun to the surface and at or near the pellet. However, it was not detectable in the fractions immediately below the surface lipids (Fig. 2F). This suggested that BicD was not simply everywhere in the gradient. Using the same antibodies for immunoflouresence (in the whole embryo) clearly demonstrated a general lack of BicD signal in the t = 3 background as compared to wild-type (Fig. 2H, I). In wild-type embryos strong punctae were observed, which were predominantly lacking in the BicD-low mutant. Some of these punctae were found close to lipid droplets (Fig. 2G). Subsequent larger volume and higher speed lipid purifications (used our previously developed protocol to purify lipid droplets) confirmed BicD’s presence in the droplet fraction. Extensive characterization of the purification protocol [20] has established that the lipid droplet fraction does not have large contaminants. The presence of BicD on the droplets is consistent with a direct role in facilitating/regulating transport. Lowered embryonic BicD levels alter individual droplet motion. To differentiate between an actual transport problem and simply a problem tethering droplets once they are delivered, we analyzed the motion of droplets with high temporal and spatial resolution. While a global analysis suggested a role for BicD in controlling the Phase III distribution of lipid droplets, altered transport in Phase II could in principle contribute to phenotypes PLoS ONE | www.plosone.org 3 November 2008 | Volume 3 | Issue 11 | e3763