BicD Regulates Transport Figure 2. Lowering BicD expression has a net effect on lipid droplet transport. A and B: Transmitted light images of developing BicDnull (t = 3) embryos indicate proper lipid droplet clearing in phase II (A), but an inability to recloud in phase III (B). C: Prior to Phase 3, during germband extension, the neutral-lipid specific dye Nile Red shows a net position of lipid droplets which is very different between wild-type and BicDnull embryos. D: During germband extension, DAPI autoflouresence of yolk vesicles does not indicate a significantly different positioning of these cargoes. Dashed lines delineate the embryos edge. E: No gross defects are observed in the MT structure of mutant embryos, as exemplified by the above image of MT immunoflouresence. Both images show microtubules passing from above, between, and below the syncytial nuclei. F: BicD localizes to the layer of lipid droplets which spin up through a sucrose gradient during centrifugation from an initial embryo lysate due to their bouyent density. Equal volumes of extracted sample were loaded per lane of the western. BicD signal can be found in the pellet, the layer immediately above the pellet (-800), and at the surface where lipid droplets coalesce. G: Confocal analysis of whole fixed embryos shows some BicD signal (green) close to droplets (red). Red signal represents the droplets specific marker LSD2. Lipid droplets generally average approximately 500 nm in size. H and I: With less magnification, standard fluorescence microscopy shows significant punctual staining of the cytoplasm in Phase I wild-type embryos (H) but not in Phase I, BicDnull (t = 3) embryos (I). Here, green is BicD, and red is the lipid droplet specific protein LSD2. J and K: Phase II DIC images show a clear difference in the number of lipid droplets near the nuclei. Dashed lines specify the position of the nuclei and red arrows are shown indicating the position of several lipid droplets. L: Automated analysis of the number of droplets observed per frame, in phase II, confirms the apparent decrease in droplets seen at the periphery of the embryo in ‘t = 1’ and ‘t = 3–4’ mutant backgrounds. doi:10.1371/journal.pone.0003763.g002 observed in Phase III. We therefore recorded and analyzed droplet motion in both phase II and III. We initially examined motion in Or-R embryos (wild-type, phase II, N = 9 embryos, n = 305 droplets tracked; phase IIB, N = 3 embryos, n = 143 droplets; phase III, N = 2 embryos, n = 45 droplets), t = 1 embryos (low BicD, phase II, N = 11 embryos, n = 52 droplets; phase IIB,N = 9 embryos, n = 18 droplets) and t = 3,4 embryos (BicD,null, phase II, N = 7 embryos, n = 19 droplets; phase IIB, N = 5 embryos, n = 12 droplets; phase III,N = 5 embryos, n = 50 droplets). Comparing wild-type motion to motion in the t = 3 background, we find that loss of BicD shortened both plus-end and minus-end run in phase II, but only minus-end runs in phase III (Fig. 3A, B). While we presume that decreased overall embryonic BicD levels result in decreased droplet-bound BicD levels (consistent with the disappearance of BicD puncta in the low-BicD embryos, Fig 2), we cannot directly show this because we cannot purify droplets from the limited number of developing mutant embryos. We also discovered a new intermediate phase of transport, phase IIb, wherein there is a large up-regulation of both plus- and minus-end directed run lengths in wild-type (Fig. 3C). Chrono- logically, this phase of transport lasts for ,10 minutes, and appears to coincide with the reversal of the net direction of transport. In the t = 3 (,null) embryos, this phase does not occur: the quantitative parameters of droplet motion in phase II t = 3 embryos are the same as for phase IIb t = 3 embryos (t-test, p = 0.56). Thus, the presence of BicD is somehow required for the regulatory changes involved in control of this set of changes in droplet motion. Intriguingly, in the t = 1 embryos where there is a small yet significant residual amount of BicD (Fig. 1E), some form PLoS ONE | www.plosone.org 4 November 2008 | Volume 3 | Issue 11 | e3763