Instead, we hypothesize that during normal wild-type develop- ment, BicD’s motor regulatory functions can be auto-inhibited by an intramolecular BicD-BicD interaction, but that the PA66 point mutation has altered this interaction. Our model (Fig. 7) is as follows. First, we hypothesize that BicD can move between two main conformations, a ‘closed’ conformation (Fig. 7a, left) and an ‘open’ conformation (Fig 7a, right). Such a range of conformations is consistent with past EM studies visualizing BicD [10] (however, we do not attempt to differentiate between the possibility that intermediate states could reflect either the modulation of time spent in the completely-open vs completely-closed states, or the possibility that an intermediate conformation could be adopted for a given period of time). Secondly, it has been previously suggested [8,12] that BicD may be capable of self-inactivation by positioning its N- and C-terminal ends near one another. In our case, we propose that when BicD is in its ‘closed’ state, it cannot regulate/ activate either the plus-end or minus-end motors, but that it is maximally able to activate such motors when in an entirely ‘open’ state. This hypothesis builds on previous observations that fragments of BicD can interact with dynein [7,8,13] and kinesin [19], but that this ability can be absent or minimized in the full- length protein. The functional relevance of such conformational changes are consistent with the observation that while the N- terminus is generally considered the dynein recruiting fragment, there are also dynamitin (dynactin) and dynein intermediate chain (D.I.C.) interacting regions of BicD at the C-terminal portion of BicD (Fig 1B). BicD Regulates Transport From our data studying the PA66 mutation, we further hypothesize that the BicD-BicD inhibitory interaction is itself actively regulated during development, and the PA66 mutation alters the regulation of the interaction (Fig 7C). Our model is that during wild type development, with normal BicD function, run- lengths are in part controlled by the amount of time BicD spends in the ‘open’ configuration–or alternatively, how ‘open’ it is (Fig. 7B). Thus, the extremely long runs in phase IIB would reflect BicD being completely ‘open’, and both phase II and phase III would reflect slightly more closed forms of BicD–or alternatively, BicD spending more time in a ‘closed’ configuration. Implicit in this model is the hypothesis that plus-end motion is more sensitive to exact BicD levels than minus-end motion: in phase II and III there is a basal level of BicD activity sufficient to achieve a fixed level of minus-end transport, but to really up-regulate minus-end transport requires the ‘completely open’ conformation found in IIB. In contrast, the moderately higher level of BicD activity in phase II relative to phase III (perhaps due to higher overall levels of the protein present on the droplet) is enough to increase plus- end motion. Such a sensitivity of plus-end motion to exact BicD levels/activity is consistent with the larger response of plus-end motion to the slightly increased BicD levels in t = 1 vs t = 3 embryos (see Fig. 3A, B). Interpreting the phenotype of BicDPA66 within this general framework suggests that the point mutation affects the regulation of the closed versus open states of BicD, but requires that whatever role BicD has in the initial recruitment of dynein (if any) is not Figure 7. A proposed model for the proper regulation of BicD and the effect of the PA66 lesion. A: Past studies have indicated that BicD has a flexible linker region between its two globular domains (Stuurman et al., 1999) and that an intramolecular interaction of the N- and C-terminal domains might represent a closed/inactive state (A, left) that can open to interact (A, right) with other regulatory components (Hoogenraad et al., 2001). B: Wild-type BicD may regulate its active state by either controlling how frequently it alternates between a completely open/active vs. a completely closed/inactive state or through an ability to take an intermediate conformation. Therefore, intermediate conformations shown for wild- type (B left, B right) are simply intended to suggest that BicD molecules are not locked in one state or the other. Yet, the open stature of BicD shown for phase IIb of wild-type (B, middle) is intended to suggest a relatively open/active state. C: The A40V lesion of BicDPA66 could affect N- to C-terminal interaction within BicD. BicDPA66 tracking results could suggest that there is an abrupt change from a closed to an open state sometime near phase IIb, followed by a clear inability down-regulate the open/active state once it has occurred, as seen in phase III (C). doi:10.1371/journal.pone.0003763.g007 PLoS ONE | www.plosone.org 10 November 2008 | Volume 3 | Issue 11 | e3763