Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex
- University of Vermont, United States
- National Heart, Lung, and Blood Institute, National Institutes of Health, United States
- Johns Hopkins University, United States
Figures
Figure 1 with 1 supplement
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 …
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Figure 1—source data 1
Dataset for Figure 1.
- https://doi.org/10.7554/eLife.36306.005
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 …
Figure 2 with 1 supplement
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 …
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 …
Figure 3 with 5 supplements
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 …
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. …
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 …
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 …
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 …
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Figure 4—source data 1
Dataset for Figure 4.
- https://doi.org/10.7554/eLife.36306.016
Figure 5 with 1 supplement
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 …
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Figure 5—source data 1
Dataset for Figure 5.
- https://doi.org/10.7554/eLife.36306.019
Figure 5—figure supplement 1
Kymograph of (left panel) a minimal dynein-dynactin-BicD2N complex, visualized with a Qdot bound to BicD2N, and (right panel), a complex of dynein, dynactin, full-length Drosophila BicD, Egl and K10 mRNA labeled with Alexa Fluor 488-UTP.
The straight vertical line at the left of both kymographs shows complex accumulation at the minus-end of the microtubule.
Figure 6
Motile properties of complexes containing one versus two Egl molecules.
(A) Schematic of the two-color experiment in which Egl is labeled with either a 565 or 655 nm QDot (figure adapted from [Reck-Peterson et al., 2018]). (B) Kymographs of dual-color runs. (Left panel) …
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Figure 6—source data 1
Dataset for Figure 6.
- https://doi.org/10.7554/eLife.36306.021
Figure 7
Recruitment of two dynein motors to the mRNP results in faster and longer runs.
(A) Schematic of the two-color experiment. Dynein was either labeled with Alexa 488 (green) or Alexa 647 (red) for the single-molecule pulldowns shown in panel B. mRNPs were formed by incubating …
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Figure 7—source data 1
Dataset for Figure 7.
- https://doi.org/10.7554/eLife.36306.023
Figure 8
Comparison of motile mRNPS reconstituted with WT dynein or dynein without LC8.
(A) SDS-PAGE of (lane 1) molecular mass markers, (lane 2) expressed WT dynein, (lane 3) dynein expressed without LC8. The identity of the bands labeled DIC and DLIC was confirmed by mass …
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Figure 8—source data 1
Dataset for Figure 8.
- https://doi.org/10.7554/eLife.36306.026
Figure 9
The mRNP predominantly contains one K10 mRNA.
(A) Schematic of the two-color mRNA experiment. mRNA was either labeled with Andy 488-UTP (green) or Andy 647-UTP (red) (figure adapted from [Reck-Peterson et al., 2018]). (B) 5.5% of moving …
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Figure 9—source data 1
Dataset for Figure 9.
- https://doi.org/10.7554/eLife.36306.028
Figure 10
A truncated Egl-leucine zipper construct supports motility in the absence of mRNA.
(A) Kymographs illustrating motion of mRNPS reconstituted from dynein-dynactin-BicD-Egl-K10 mRNA, dynein-dynactin-BicD-Eglzip, dynein-dynactin-BicD-Egl, or dynein-dynactin-BicD. (B) Run frequency …
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Figure 10—source data 1
Dataset for Figure 10.
- https://doi.org/10.7554/eLife.36306.030
Videos
Video 1
Low-resolution 3D map of negative stain EM data (related to Figure 3).
The video shows the size comparison of the apparent loop (EM volume depicted in gray mesh) with existing structures for parts of the YFP-BicD-Egl complex. The map has not been validated, and serves …
Video 2
mRNPs with two dyneins move faster and longer (related to Figure 7).
The DDBE plus K10 mRNA complex containing two dimeric dyneins (yellow due to colocalization of a red and green Qdot) moved 12.2 µm in 20.4 s at a speed of 0.6 µm/s on a microtubule track …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | Alexa Fluor 488–5-UTP | Molecular Probes | C11403 | |
| Chemical compound, drug | Andy Fluor 488-X-UTP | GeneCopoeia | C410A | |
| Chemical compound, drug | Andy Fluor 647-X-UTP | GeneCopoeia | C418A | |
| Chemical compound, drug | Rnase Inhibitor | Promega | N261B | |
| Chemical compound, drug | Q-dot 525 streptavidin conjugate | Invitrogen | Q10141MP | |
| Chemical compound, drug | Q-dot 565 streptavidin conjugate | Invitrogen | Q10131MP | |
| Chemical compound, drug | Q-dot 655 streptavidin conjugate | Invitrogen | Q10121MP | |
| Chemical compound, drug | SNAP-Surface Alexa Fluor 488 | New England BioLabs | S9129S | |
| Chemical compound, drug | SNAP-Surface Alexa Fluor 647 | New England BioLabs | S9136S | |
| Chemical compound, drug | SNAP-Biotin | New England BioLabs | S9110S | |
| Chemical compound, drug | Ribonucleic acid, transfer from Escherichia coli | Sigma-Aldrich | R1753 | |
| Chemical compound, drug | Tubulin protein (X-rhodamine): bovine brain | Cytoskeleton, Inc | TL620M-A | |
| Chemical compound, drug | paclitaxel | Cytoskeleton, Inc | TXD01 | |
| Commercial kit | RiboMAX Large Scale RNA Production Systems | Promega | P1280 | |
| Recombinant DNA | pDyn1 (SNAPf-His-ZZ-LTLT- DYNC1H1 in pACEBac1) (Homo sapiens) | Schlager et al. (2014) | NCBI:NP_001367.2 | Expression plasmids for dynein in Sf9 cells. See details in Materials and methods. |
| Recombinant DNA | pDyn3 (ZZ-SNAPf-DYNC1H1, DYNC1I2, DYNC1LI2, DYNLT1, DYNLRB1,DYNLL1 in pDynBac1) (Homo sapiens) | Schlager et al. (2014) | NCBI:NP_001367.2 | Expression plasmids for dynein in Sf9 cells. See details in Materials and methods. |
| Recombinant DNA | dynein nBiotin tag (Homo sapiens) | This paper | NCBI:NP_001367.2 | Expression plasmids for dynein in Sf9 cells. See details in Materials and methods. |
| Recombinant DNA | Bicaudal D, isoform A (Drosophila melanogaster) | This paper | NCBI:NP_724056.1 | Expression plasmids for BicD, YFP-BicD in Sf9 cells, and BicDCC1 in E. coli. see details in Materials and methods. |
| Recombinant DNA | Bicaudal D homolog 2 isoform 2 (Homo sapiens) | This paper | NCBI:NP_056065.1 | Expression plasmids for BicD2N in E. coli. See details in Materials and methods. |
| Recombinant DNA | Egalitarian (Drosophila melanogaster) | This paper | NCBI:AAB49975.2 | Expression plasmids for Egl in Sf9 cells, and Egl-ZIP in E. coli. See details in Materials and methods. |
| Recombinant DNA | K10 mRNA (Drosophila melanogaster, f) | This paper | NCBI:NM_058143.3 | Expression plasmids for K10 mRNA, K10 no zip mRNA. See details in Materials and methods. |
| Recombinant DNA | β-actin mRNA (Rattus norvegicus) | This paper | NCBI:NM_031144.3 | Expression plasmids for b actin mRNA. See details in Materials and methods. |
| Recombinant DNA | ASH1 mRNA (Saccharomyces cerevisiae) | Sladewski et al. (2013) | NCBI:NM_001179751.1 | Expression plasmids for Ash1 mRNA. See details in Materials and methods. |
| Recombinant DNA | kinesin G235A (Mus musculus) | This paper | NCBI:NM_008449.2 | Expression plasmids for rigor kinesin in E. coli. See details in Materials and methods. |
| Biological sample | dynein - dynactin | Bovine brain | See details in Materials and methods. | |
| Biological sample | tubulin | Bovine brain | See details in Materials and methods. | |
| Software, algorithm | Nikon ECLIPSE Ti microscope | Nikon | ||
| Software, algorithm | Nikon NIS Elements | Nikon | ||
| Software, algorithm | Andor EMCCD Camera | Andor Technology USA | ||
| Software, algorithm | Prism | GraphPad | v7; RRID:SCR_002798 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.36306.031
