BicD Regulates Transport Figure 3. Droplet distribution is altered due to BicD-induced changes in Droplet motion. A: BicD contributes to correct minus-end motion. In the t = 3,4 background, minus-end directed run lengths are shorter in all three phases under consideration. While a clear run length peak is present in phase IIb of wild-type, there is no such peak in the null. B: Comparison of plus-end directed run lengths shows that BicD affects this direction of transport as well, although, not in phase III (B). Note the difference between t = 1 and t = 3 plus-end run lengths (t-test, p = 0.049) in phase II (A). A similar difference is not apparent in the minus-end direction (t-test, p = 0.56) of transport (B). C and D: The net reversal of directionality seen to occur in phase IIb for wild-type (C) does not occur in the mutant (D). doi:10.1371/journal.pone.0003763.g003 of the Phase IIRIIb transition occurs, but its form is different: in the t = 1 embryos, the plus-end run lengths go down instead of up (Fig. 3). This is statistically obvious for plus-end runs (t-test, p = 5.361024; Fig. 3B), and there is a hint that this might be true for minus-end motion as well (Fig. 3A), although we cannot be certain because the effect in the minus-end direction is small, and within uncertainty (t-test, p = 0.12). Regardless, the decrease in plus-end run lengths during the phase II to IIb transition of the t = 1 embryos (t-test, p,0.04), is in the opposite direction from the change seen in wild-type where run-lengths increase (t-test, p,0.04), suggesting that the exact levels of BicD present may play an important role during this transition under normal circumstances in the wild-type. Supporting the hypothesis that plus-end transport may be quite sensitive to BicD levels, we note that in phase II, minus-end motion in the t = 1 and t = 3 backgrounds is approximately the same (t-test, p = 0.56; Fig. 3A), but the small amount of additional BicD present in the t = 1 embryos is enough to significantly improve Phase II plus-end motion above that found in the t = 3 embryos (t-test, p = 0.049; Fig. 3B). Droplet-bound BicD levels are temporally changing, and regulated Since the t = 1/t = 3 experiments suggested that BicD levels may be important, we investigated whether droplet-bound BicD levels changed in conjunction with normal developmental changes in droplet motion. To do this, rather than purifying droplets from embryos in a 4-hour collection, we used a previously developed approach [16,20] to do temporally staged purifications, enabling us to investigate whether there were any changes in droplet-bound BicD levels as a function of time. To our surprise, levels of BicD on the droplet decreased precipitously from phase 1 to 3 (Fig. 4A). This result was repeated multiple times and sample volumes per lane were normalized for total protein each time. Droplet-bound BicD levels could be actively controlled, or could passively reflect changing embryonic levels of BicD. To differen- tiate between these possibilities, we did temporally controlled collections, as described above with respect to droplet purifica- tions. However, rather than purifying the droplets, we simply looked at overall embryonic levels of BicD. We found (Fig. 4B) that embryonic BicD levels are relatively stable during this period, PLoS ONE | www.plosone.org 5 November 2008 | Volume 3 | Issue 11 | e3763