recruiting dynein to the cargos. These past studies are important, but rely heavily on over-expression studies, and/or expression of truncated forms of the protein, and so leave open the question of whether BicD levels or activity are changed as part of the usual regulatory process. In addition, even accepting that BicD is important as a dynein recruiter, there has been open the question of whether BicD plays any continuing regulatory roles, distinct from making sure that the transport complex is appropriately set up. Our studies have investigated these questions within the context of bi-directional lipid droplet motion in Drosophila embryos. BicD appears not to function only as a static dynein tether Our data is consistent with the hypothesis that BicD plays a role in recruiting dynein to the cargo: loss of BicD function leads to many fewer droplets present in the embryo, and hence fewer moving droplets, consistent with a role for BicD in recruiting dynein to droplets during embryogenesis, allowing them to be subsequently delivered into the egg. However, some dynein recruitment is BicD-independent, because the same number of droplets make it into the embryo in the t = 1 and t = 3 embryos (and approximately the same number of droplets are moving in these two backgrounds) as determined from tracking analysis, in spite of the 66 difference in BicD levels, and the fact that in the t = 3 embryos BicD levels have been reduced ,3756 relative to the wildtype, so it seems unlikely that there is still enough BicD remaining to be functional. This finding is consistent with past work investigating BicD’s role in mRNA transport, which also concluded that BicD was not absolutely essential to recruit dynein [14]. Although our favored interpretation is that BicD plays a role in dynein recruitment, past work [21,22] suggested that in ovaries of BicD-mutant mothers, the microtubule cytoskeleton can be impaired, resulting in improper transport of components into the developing embryos. Such disorganization could contribute as well to the apparent decrease in droplets present in the BicD-mutant embryos. Overall, although there are fewer droplets apparently present in the mutant embryos, the percentage of droplets present that actually move is approximately the same for all the genotypes, at ,10%. We also note that there remains the possibility that some of the apparent decrease in embryonic droplets reflects increased basal droplet localization, to a central area of the embryo where droplet quantitation is difficult. Nonetheless, numerous lines of evidence suggest that either a rather small portion of the available BicD tethers the dynein, or that whatever role BicD plays in dynein recruitment occurs in a temporally limited fashion. Loss of almost all the droplet-bound BicD (Fig. 4A) due to normal developmental regulation does not result in any loss of dynein from the droplets, as judged either by western blot, or stalling forces (we’ve shown previously[23,24] that stalling force is roughly proportional to the number of engaged motors), or mean minus-end travel distances. A limited duration for BicD to help assemble the motor complexes is consistent with previous mRNA studies that suggest that the nature of the complexes moving the mRNA particles is sensitive to BicD levels when the complexes are formed, but not afterward [14]. BicD contributes to dynamic regulation of droplet motion We find that approximately the same number of droplets are moving in both of the very low-BicD backgrounds (Fig. 6F), although western blotting of embryos shows that there is ,66 more BicD in the t = 1 background relative to the t = 3 BicD Regulates Transport background. This suggests that at such low levels of BicD, BicD can no longer play a significant role in recruiting dynein to the droplets (if it could, one would expect many more moving droplets in the t = 1 embryos). Thus, we hypothesize that there is a BicD- independent pathway for some dynein to get on to the droplets. Assuming that this hypothesis is true, i.e. that the levels of BicD present in both the t = 1 and t = 3 embryos are indeed too low to facilitate dynein recruitment, the observation that motion is altered between the two suggests that BicD likely has an additional regulatory role, independent of simple dynein recruitment, and that this regulatory role is affected by the amount of BicD present on the cargo. The existence of this dynamic regulatory role is further confirmed by the interesting phenotype of motion in the PA66 background, where the mutant form of the protein allows a dramatic change in droplet motion between phase II and IIb, but the subsequent down-regulation of transport expected between phase IIB and III fails to occur. A model for how the point mutation affects BicD’s regulatory role is described below. BicD plays a role in kinesin-mediated travel In addition to the evidence presented above supporting roles for regulation of minus-end transport, our data shows that loss of BicD function leads to altered plus-end motion, suggesting a new role for BicD: regulating plus-end transport. Our past studies suggest a strong coupling between plus-end and minus-end motion, so one question is whether the observed effects are indirect, and reflect a primary role in minus-end transport, with the altered minus-end transport resulting in plus-end transport. While we cannot be certain, we believe it is more likely that BicD plays a role in direct regulation of plus-end transport. First, recent work found that the middle portion of BicD interacts with kinesin via 2-hybrid [19]. When published, the functional significance of the interaction was unknown, but our data supports the hypothesis that it may be important. Our recent work[24]establishes that plus-end droplet motion is driven by kinesin-1, so it is unlikely that the effects of BicD on plus-end motion are simply due to its effects on dynein, although recent work suggests that this may be the case for some mRNA transport[25]. Second, the large phase IIb run length peak is absent in the t = 3 null, consistent with the hypothesis that BicD can acutely up-regulate both directions of transport. Third, we noted above that plus-end transport appears to have higher dosage sensitivity than minus-end transport to BicD levels (Fig. 3A, B). Regardless of the exact mechanism, our studies show that BicD plays an important role in regulating the relative contribution of plus-end versus minus-end motion. Although run lengths are very long in phase III of the PA66 background, a reversal of the net direction of transport is not observed (Fig. 6J). Thus, the mutant analysis indicates that BicD’s role in balancing plus-end versus minus-end motion is functionally separable from its role in facilitating overall upregulation of transport, with the resulting long runs in each direction in Phase IIB (Fig. 6H, I). A model for BicD function, incorporating findings from the PA66 point mutation BicDPA66 is interesting in that the altered protein maintains the ability to up-regulate the high phase IIb run lengths, yet cannot regulate net directionality. If phase IIb run length up-regulation requires BicD, why are run lengths in phase II of the BicDPA66 background similar to the t = 3 null? In principle, BicD could be absent from the droplets in phase II and then rapidly accumulate at the onset of phase IIb, but this seems unlikely given that such a recruitment of BicD to the droplets in Phase IIB would go counter to the continued drop in droplet-bound BicD levels which usually occur during development. PLoS ONE | www.plosone.org 9 November 2008 | Volume 3 | Issue 11 | e3763