severely altered by the mutation (since there are more moving droplets in the BicDPA66 embryos than in the t = 1 or t = 3 embryos). We hypothesize that in contrast to normal Phase II BicD which spends some time partly open, phase II BicDPA66 is unable to respond to regulatory signals that keep it partly open. It would therefore be locked completely in the ‘closed’ state, and be unable to properly activate the motors. It thus acts as a ‘null’ mutant in phase II (i.e. the tracking phenotype is the same as for the t = 3 background). Then, for phase IIb, the mutant protein responds appropriately to a second regulatory event, that induces maximal opening of BicD in phase IIb. Consequently, the phenotype disappears, and phase IIb motion is excellent; there is the suggestion that runs may be even longer than wild-type in the PA66 background. Finally, just as the mutant was unable to respond to whatever signal was present in phase II (to keep it partly open), in phase III it is unable to respond to the signal to partly close up, and remains fully open. Functionally, this causes BicDPA66 phase III run lengths to be quite similar to those of phase IIB, i.e. the expected significant decrease in phase III run lengths fails to occur. In conclusion, we believe that the BicD N-terminus/ C-terminus interaction is regulated to control the amount that BicD increases plus-end and minus-end transport, and that this regulation is altered by the A40V mutation. Our data shows that BicD function is essential for the ability to regulate bi-directional lipid droplet motion. BicD activity alters both plus-end and minus-end transport, and can play a role in balancing the relative contributions of each to allow regulated net transport. More than simply a physical attachment between dynein and the cargo, BicD plays interesting roles in regulating motion. It is interesting to speculate that BicD could do this by altering or facilitating the interaction of dynactin with dynein and possibly kinesin. Such a hypothesis is consistent with our past work and that of others implicating dynactin in coordinating opposite motors [26,27], with the finding that for mRNA particles dynactin modulates dynein-mediated movement [25], and with the previous findings discussed above that BicD can bind both Dynactin and Dynein, thus in principle allowing it to act as an intermediary between the two. However, the extent that this occurs—and if so the mechanistic details of such a process—remains for future work to determine. Analysis of the BicDPA66 point mutant demonstrates that BicD’s dual abilities—to facilitate up-regulation of transport, and to facilitate switching the net direction of transport—are both regulated and separable. Clearly, a great deal of future work will be required to understand these functions at a molecular level, but the model proposed here should be helpful in guiding some of these new studies. Materials and Methods Fly Stocks and Crosses Oregon-R was the wild-type stock. BicDr6/Df(2L)TW119; P[w+ hsp70 BicD]-94/+ served as our for purposes of particle tracking. Our BicDr6 and Df(2L)TW119/CyO stocks were obtained from the laboratory of Beat Suter. The associated heat shock rescue protocol is based on that described in Swan and Suter 1996. Our slightly modified version involved one hour, once a day, 37u C heat shocks from early larval stages until adulthood. Heat shock rescued BicDnull females and their embryos were considered ‘t = 1’ one day after and ‘t = 3/4’ three/four days after cessation of the heat-shock regimen. The HA-tagged BicD transgene inserted on the first chromosome expresses approximately 12% of wild- type levels as previously described [11]. Therefore, a HA40::BicD/ +; BicDnull/Df female expressed approximately 6% of wild-type BicD Regulates Transport BicD levels. The HA40:BicD stock, H2D stock, and H3D stock were provided by the lab of Ruth Steward. Other stocks used in this study are publicly available on FlyBase (http://flybase.bio. indiana.edu). For the terminal cross resulting in embryos to be analyzed, Wild-type OrR males were used to fertilize the females, i.e. OrR males were crossed to (a) heat-shock rescue (null) females (i.e. BicDr6/Df119; Phs.BicD/+), to (b) ‘‘PA66 females’’ (HA40::BICD/+; PA66/PA66), and were also used for (c) all reclouding rescue experiments. Embryo Staging Preliminary one hour embryo pre-collections were collected and discarded in order to eliminate fertilized eggs that females flies may have been storing. Timed phase specific purifications were based on one hour collection windows. Phase zero collections were stopped (e.g. lysed, fixed, etc.) immediately after collection. Phase I, II, and III collections were aged 1.5, 2.5, and 3.5 hours respectively following the one hour collection and then stopped. In contrast, embryos squashed for particle tracking were staged by eye. Phase II tracking was staged by identifying the point at which the cellularizing plasma membrane had moved halfway down the length of the syncytial nuclei. Phase IIb was defined as beginning when the membrane reached the basal edge of the nuclei. Phase III was defined as beginning at the initial dorsal movement of the pole cells which occurs early in embryogenesis. Isolation of Lipid Droplets by Subcellular Fractionation Lipid droplets were purified as described previously (Cermelli et al., 2006). In brief, embryo lysates were loaded beneath a sucrose gradient solution and ultracentrifuged. Afterwards, the buoyant lipid droplets were collected from the top of the gradient. Isolated lipid droplets were then solublized in SDS-containing buffer for subsequent SDS-PAGE analysis. The step gradient western seen in figure 2f is based on a scaled down version of the above mentioned purification protocol. Particle Tracking and Statistical Analysis Droplet motion was recorded as previously described [28]. Particle tracking was performed using automated analysis of each video frame. Runs were defined as ending only when a reversal in the direction of transport was observed. Pauses were included as part of a continuous run if a reversal was not identified. We used custom video tracking software to analyze our data (details to be published elsewhere). Briefly, we determined the location of lipid droplets in each video frame via template matching [29] and then used these records of droplet positions (versus frame number) to construct motion tracks for each particle. Because lipid droplets are extended objects in our videos (,500 nm in diameter), tracks could be unambiguously assembled based on particle proximity in subsequent frames. Tracks were then filtered to identify droplets which moved extended distances over many frames along a line perpendicular to the edge of the embryo [16]. Here, as a quantitative measure of overall motion of droplets, for each field of view (for all movies of a given genetic background) we also report the ratio of the droplets moving directionally (1 um minimum travel length) to the total number of detected droplets (no minimum travel length requirement). This latter quantity was determined as above, however no filtering was performed on the tracks. MT staining and Lipid Droplet Immunoflouresence Microtubule staining of embryos involved dechorionating in 50% bleach, rinsing in water and fixing in a mix of heptane and PLoS ONE | www.plosone.org 11 November 2008 | Volume 3 | Issue 11 | e3763