Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex

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,…

Figure 1—figure supplement 1.. SDS-PAGE gels of BicD constructs and tissue-purified bovine brain dynein and dynactin. (A) (lane 1) Drosophila BicDCC1, (lane 2) full-length Drosophila BicD, (lane 3) human BicD2N, and (lane 4) molecular mass markers. 4–12% SDS-PAGE gel, MOPS buffer. (B) dynein and dynactin purified from bovine brain, and molecular mass markers. 4–12% SDS-PAGE gel, MOPS buffer.

Figure 2.. Electron micrographs of the auto-inhibited full-length YFP-BicD. (A) A montage of negatively stained images showing (row 1) the most common ‘b’ orientation, (row 2) a less common ‘d’ orientation, (row 3) the range over which the molecule can flex, (row 4) compact molecules, and (row 5) rare open molecules. (B) Global average of all aligned YFP-BicD images. (C) Example class averages of BicD with classification focused on the loop. (D) One YFP-BicD molecule illustrating the interpretation of the EM image, with the length of the long straight section indicated. (E) Example class averages of YFP-BicD with classification focused on the protruding YFP-CC1 region. See Figure 2—figure supplement 1 for full classifications. (F) Pulldown showing that CC1 interacts with CC2-CC3 but not CC2. Scale bars = 20 nm.

Figure 2—figure supplement 1.. Image processing scheme for YFP-BicD EM data. Following initial alignment of the full dataset (n = 4205), images were classified into 100 classes using a variance mask. Classes in similar orientations were selected (based on the appearance of class averages and class variances, asterisks show selected classes). Particles from selected classes were re-aligned (n = 1513). Aligned images were classified into 50 classes using masks around the loop or the protruding YFP-CC1 region. Scale bars = 20 nm.

Figure 3.. Electron micrographs of the YFP-BicD-Egl and YFP-BicD-Egl-mRNA complex. (A) Montage of images showing that BicD retains the auto-inhibited looped conformation in the presence of bound Egl. A schematic of Egl (below) shows that the N-terminal domain binds BicD, the C-terminus binds dynein light chain (LC8), and mRNA binding is mediated through a large number (~800) of amino acids. (B) Montage of images showing that YFP-BicD no longer retains the auto-inhibited looped conformation in the presence of bound Egl and mRNA (K10min). Global and class averages are shown below the montage. (C) Average of all YFP-BicD-Egl molecules. (D) Example class averages of YFP-BicD-Egl with classification focused on the loop. (E) Example class averages of YFP-BicD-Egl with classification focused on the protruding YFP-CC1 region. (F) Example class averages of YFP-BicD-Egl with classification focused adjacent to the loop, to reveal the globular domain of Egl. (G) Co-alignment and classification of YFP-BicD and YFP-BicD-Egl. Upper row shows the average of YFP-BicD and YFP-BicD-Egl images (after an initial alignment, classification and selection of particles in similar orientations). Difference maps are…

Figure 3—figure supplement 1.. SDS-PAGE gels of Egl. (lane 1) Molecular mass markers, (lane 2) BicD co-expressed with Bio-tagged Egl, (lane 3) Egl expressed alone. 4–12% SDS-gel, MOPS buffer.

Figure 3—figure supplement 2.. EM of other conditions imaged in this study. (A) Montage showing example raw images of mRNA (K10min). Global average and example class averages are shown below the montage (n = 2039). (B) Montage showing example raw images of YFP-BicD-mRNA. Global average and example class averages are shown below the montage (n = 1393). (C) Montage showing example raw images of BicD-Egl. Global average and example class averages are shown below the montage (n = 3697). (D) Montage showing example raw images of BicD-Egl-mRNA. Global average and example class averages are shown below the montage (n = 3526).

Figure 3—figure supplement 3.. Fields of view of all conditions imaged in this study. Scale bars = 50 nm.

Figure 3—figure supplement 4.. Image processing scheme for YFP-BicD-Egl EM data. Following initial alignment of the full dataset (n = 4117), images were classified into 100 classes using a variance mask. Classes in similar orientations were selected (based on the appearance of class averages and class variances, asterisks show selected classes). Particles from selected classes were re-aligned (n = 1488). Aligned images were classified into 50 classes using masks around the loop, the protruding YFP-CC1 region or the region adjacent to the loop, in order to reveal the Egl globular domain. Scale bars = 20 nm.

Figure 3—figure supplement 5.. Other classifications used in this study. (A) Classification of BicD-Egl using a mask adjacent to the loop to reveal the globular domain of Egl. (B) Classification of BicD-Egl using a mask around the loop. (C) Classification of BicD-Egl using a mask around the protruding CC1 region. (A), (B) and (C) are classifications of re-aligned images (n = 1765) following an initial alignment (n = 3697) and selection of classes in the most common orientation. (D) Classification of BicD-Egl-mRNA (n = 3526). Scale bars = 20 nm. (E) Classification of YFP-BicD-Egl-mRNA (n = 3345). (F) Classification of combined YFP-BicD and YFP-BicD-Egl datasets (most common orientation only). Central row shows difference images between the two datasets. Scale bars = 20 nm.

Figure 4.. K10 mRNA is needed for BicD-Egl to recruit dynein-dynactin for motility. (A) Single-molecule pulldowns of YFP-BicD (green) and Qdot-Egl (red) by dynein-dynactin (DDBE) bound to microtubules (blue) in the absence or presence of K10 mRNA. (B) Quantification of the single-molecule pulldown data showing the normalized number of colocalized BicD-Egl complexes bound to dynein-dynactin per µm microtubule length in the absence of mRNA (DDBE), in the presence of K10 mRNA (DDBE + K10), or K10 without the TLS zip code (DDBE + K10 no zip). (C) Normalized run frequencies (per μM dynein per μm microtubule length per time) of motile mRNPs. (Bars, left to right) Little movement of labeled K10 mRNA in the absence of either BicD or Egl. Few events are also observed for mRNPs lacking mRNA (movement was visualized with a Qdot bound to Egl). Fully reconstituted mRNPs with K10 mRNA have the highest run frequency, while mRNPs reconstituted with K10 mRNA lacking the TLS zip code showed reduced run frequencies. Two unrelated mRNA constructs, S. cerevisiae ASH1 mRNA and mammalian β-actin mRNA showed run frequencies similar to that of K10 mRNA lacking the TLS zip code. Error bars, sem, n ≥ 4 movies per…

Figure 5.. The motile properties of the fully reconstituted mRNP are similar to dynein-dynactin complexes reconstituted with Drosophila DDBCC1 or mammalian BicD2N. (A) Kymograph of (left panel) a minimal dynein-dynactin-BicDCC1 (DDBCC1) complex, and (right panel) a complex of dynein, dynactin, full-length Drosophila BicD, Egl and K10 mRNA, visualized with a Qdot on BicD (B) Speed distributions of DDBE + K10 mRNA (red circles) (0.42 ± 0.22 μm/s, n = 505) and DDBCC1 (blue squares) (0.43 ± 0.26 μm/s, n = 516, p=0.25, t-test, mean ± SD). (C) Run length distributions of DDBE + K10 mRNA (red circles) (6.4 ± 0.25 μm, n = 71) and DDBCC1 (blue squares) (5.3 ± 0.17 μm, n = 66, p=0.8, Kolmogorov–Smirnov test, mean ±SE of the fit). (D) Speed distributions of DDBE + K10 mRNA (red circles) (0.45 ± 0.21 µm/s, n = 1126) and DD(BicD2N) (blue squares) (0.35 ± 0.19 µm/s, n = 1147; p<0.05, t-test, from 50 representative run trajectories for each condition, mean ± SD). (E) Run length distributions of DDBE + K10 mRNA (red circles) (7.2 ± 0.5 μm, n = 142) and DD(BicD2N) (blue squares) (5.4 ± 0.7 μm, n = 137, p=0.053, Kolmogorov–Smirnov test, mean ± SE of the fit). See Figure 5—source data 1.…