Figure 1. Full-length BicD is auto-inhibited and does not bind dynein-dynactin.
(A) Schematic of BicD constructs. Full-length Drosophila BicD contains three coiled-coil (CC) regions that are designated CC1, CC2, and CC3 (red). CC2 and CC3 are each interrupted by non-coiled-coil regions (gray). Regions predicted to form coiled-coil domains were determined from Paircoil2 analysis (McDonnell et al., 2006). Truncated versions of Drosophila BicD (BicDCC1) and mammalian BicD2 (BicD2N) are also shown. CC1 is the minimal fragment that activates dynein-dynactin, but does not bind cargo adaptor proteins, which occurs via CC3. (B) Single-molecule pulldowns on microtubules (red) show that dynein-dynactin associates with BicDCC1 (green) but not with full-length Drosophila BicD (green) in the presence of AMP-PNP (upper panels). Controls show that the two BicD constructs alone do not bind non-specifically to microtubules (lower panels). BicD was visualized with a 525 nm-streptavidin Qdot bound to an N-terminal biotin tag on BicD. (C) Quantification of the number of dynein-dependent associations of complexes containing Drosophila BicDCC1 (blue) versus full-length Drosophila BicD (red) to microtubules, normalized to microtubule length and dynein concentration (average ± SEM from 5 to 15 fields). Controls for non-specific binding are also quantified. (D) Quantification of the number of dynein-dependent associations of complexes containing mammalian BicD2N (cyan) or full-length Drosophila BicD (red) to microtubules, normalized to microtubule length and dynein concentration (average ± SEM from 5 to 15 fields), from independent experiments. See Figure 1—source data 1.
Figure 1—figure supplement 1. SDS-PAGE gels of BicD constructs and tissue-purified bovine brain dynein and dynactin.
