Dominant spinal muscular atrophy linked mutations in the cargo binding domain of BICD2 result in altered interactomes and dynein hyperactivity
- Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, United States
Figures
Figure 1 with 1 supplement
Wild-type BICD2 interactome.
(A) Schematic of human BICD2, indicating the three coiled-coil domains and sites of interaction with dynein/dynactin, kinesin-1, and cargo. (B) Model of BICD2 binding to dynein/dynactin in a cargo-dependent manner. (C, C’) A volcano plot indicating proteins that were enriched with BICD2-mTrbo in comparison to RFP-mTrbo. The dashed line along the x-axis indicates a fold enrichment of 2, whereas the dashed line along the y-axis indicates a p value of 0.05. Specific interacting proteins were considered those that were enriched at least twofold with BICD2-mTrbo and had a p value of at least 0.05. These candidates are shown in the zoomed-in image in C’. (D) Known BICD2 interacting proteins, components of the dynein motor, and components of the HOPS complex that were specifically enriched with BICD2-mTrbo are indicated. (E) A cellular component GO analysis of the BICD2 interactome.
Figure 1—figure supplement 1
HEK cells expressing the indicated constructs were transfected with a plasmid expressing GTP-locked RAB6A (GFP-RAB6A Q72L).
Lysates were prepared, and a co-immunoprecipitation experiment was carried out using V5 trap beads. The co-precipitating proteins were analyzed using the indicated antibodies. GFP-RAB6A Q72L specifically co-precipitated with full-length BICD2.
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Figure 1—figure supplement 1—source data 1
Original files for western blots displayed in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
PDF file for western blots displayed in Figure 1—figure supplement 1 with the bands marked.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
BICD2 interacts with components of the HOPS complex in vivo.
(A) A co-immunoprecipitation experiment was performed with HEK cells expressing the indicated constructs. The lysates were incubated with V5 trap beads to precipitate the tagged proteins. The co-precipitating proteins were analyzed using western blotting with the indicated antibodies. VPS41 specifically co-precipitates with BICD2_wt. Minimal binding was observed with RFP-mTrbo or a BICD2 construct lacking the cargo binding domain. (B) A similar co-precipitation experiment was set up as in panel A. The co-precipitating proteins were analyzed by blotting using antibodies against VPS16, VPS18, and the V5 epitope. VPS16 and VPS18 co-precipitate specifically with BICD2_wt-mTrbo. (C) A schematic of the BICD2 constructs used in the binding experiment. (D) Cells expressing either RFP-mTrbo, BICD2_delCC3-mTrbo (lacking the cargo binding domain), BICD2_wt-mTrbo (full length BICD2) or BICD2_CC3-mTrbo (just the cargo binding domain) were used to examine the interaction with VPS41. Lysates were incubated with streptavidin beads to precipitate biotinylated proteins. The precipitated proteins were analyzed by blotting using the indicated antibodies. Although RANBP2 interacts with the isolated cargo binding domain of BICD2, VPS41 does not.
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Figure 2—source data 1
Original files for western blots displayed in Figure 2A, B and D.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig2-data1-v1.zip
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Figure 2—source data 2
PDF file for western blots displayed in Figure 2A, B and D with the bands marked.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig2-data2-v1.zip
Figure 2—figure supplement 1
Characterization of the interaction between BICD2 and HOPS complex components.
(A) HEK cells expressing the indicated constructs were transfected with either a control siRNA or an siRNA targeting endogenous BICD2. Three days after the transfections, lysates were prepared, and a co-immunoprecipitation experiment was carried out using V5 trap beads. The co-precipitated proteins were analyzed by western blotting. RANBP2 and VPS41 co-precipitate with BICD2-wt_TurboID in cells that are depleted of endogenous BICD2. (B). Lysates were prepared from HEK cells epxpressing the indicated constructs, and biotinylated proteins were purified using streptavidin beads. The precipitated proteins were analyzed by blotting using antibodies against VPS16 and VSP18. Both proteins were able to interact with full-length BICD2 but not the isolated cargo binding domain. (C) Lysates were prepared from HEK cells expressing the indicated constructs and biotinylated proteins were purified using streptavidin beads. The precipitated proteins were analyzed by blotting using antibodies against RANBP2 and VPS41. Both proteins interact with full-length BICD2 but show a reduced interaction with BICD2_delCC1. (D) HEK cells expressing the indicated constructs were transfected with either a control siRNA or an siRNA targeting endogenous VPS41. Three days after transfection, the biotinylated proteins were purified using streptavidin beads and analyzed by western blotting using antibodies against VPS16 and VSP18. Both proteins retain their interaction with BICD2 in cells depleted of endogenous VPS41.
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Figure 2—figure supplement 1—source data 1
Original files for western blots shown in Figure 2—figure supplement 1A–D.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
PDF files for the western blots shown in Figure 2—figure supplement 1A–D with the bands marked.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig2-figsupp1-data2-v1.zip
Figure 3 with 3 supplements
Role of BICD2 in localization of GFP-VPS41 and LAMP1 vesicles.
(A–C) HeLa cells were transfected with either a control-siRNA (A), an siRNA targeting dynein heavy chain (B), or an siRNA targeting BICD2 (C). Two days after the siRNA transfection, the cells were transfected with a plasmid encoding GFP-VPS41. The next day, the cells were fixed and processed for immunofluorescence using an antibody against GFP. The cells were counterstained with DAPI (cyan) and Phalloidin (gray). Depletion of DHC results in an outward spreading of GFP-VPS41 vesicles, whereas depletion of BICD2 results in more perinuclear clustered vesicles. (D, E) The distance of GFP-VPS41 vesicles relative to the nucleus was determined and plotted. Vesicles present within 10 µm of the nucleus are shown in D, and those present at a distance greater than 10 µm are shown in panel E. (F–H) HeLa cells were transfected with the indicated siRNAs. Three days later, the cells were fixed and processed for immunofluorescence using an antibody against LAMP1. As with GFP-VPS41 vesicles, depletion of DHC resulted in peripheral vesicles, whereas depletion of BICD2 resulted in perinuclear clustering of LAMP1 vesicles. (I–K) HeLa cells were transfected with either a control siRNA (I, J) or an siRNA targeting KIF5B (K). Two days later, the cells were transfected with a plasmid encoding either GFP (I) or BICD2-mNeon (J, K). The cells were fixed on day 4 and processed for immunofluorescence using an antibody against LAMP1. The cells were counterstained with DAPI. Overexpression of BICD2 results in the peripheral spreading of LAMP1 vesicles. This phenotype was reversed upon knocking down KIF5B. (L–M) The distance of LAMP1 vesicles relative to the nucleus was determined and plotted. Vesicles present within 10 µm of the nucleus are shown in L, and those present at a distance greater than 10 µm are shown in panel M. The signal for GFP-VPS41 and LAMP1 is displayed using the ‘red hot’ LUT in FIJI. The scale bar is 20 µm. A one-way ANOVA was used for the quantifications shown in panels D, E, L, and M with the values compared to the mean of BICD2_wt. ns = not significant, *, p≤0.05, ***, p<0.001.
Figure 3—figure supplement 1
Phenotypes associated with knock-down and over-expression of BICD2.
(A) Lysates were prepared from cells transfected with the indicated siRNA. The lysates were analyzed by blotting using antibodies against DHC, BICD2, and alpha-Tubulin. The siRNAs were capable of depleting the target protein. (B) Lysates were prepared from cells transfected with two different siRNAs targeting endogenous BICD2. Both siRNAs were capable of depleting BICD2. (C) Lysates were prepared from cells transfected with either a control siRNA or an siRNA targeting KIF5B. The kif5b siRNA was capable of efficiently depleting KIF5B protein. (D) Representative image of HeLa cells showing the localization of GFP-VPS41 vesicles in cells transfected with bicd2 siRNA-2. (E–F) Representative images of HeLa cells transfected with either a control siRNA (E, E’) or bicd2 siRNA-1 (F, F’). The cells were processed for immunofluorescence using an antibody against GM130, a Golgi marker (red to white LUT). The cells were also counterstained with DAPI (cyan). Depletion of BICD2 results in fragmentation of the Golgi. (G–H) Representative images of HeLa cells transfected with either a control siRNA (G, G’) or bicd2 siRNA-1 (H, H’). The cells were processed for immunofluorescence using antibodies against Cyclin B (a G2 cell cycle marker, green) and Pericentrin (a centrosome marker, red). The cells were also counterstained with DAPI (cyan). Depletion of BICD2 results in mislocalization of the centrosome away from the perinuclear area. (I–K) Representative images of HeLa cells transfected with either a control siRNA (I), bicd2 siRNA-1 (J) or bicd2 siRNA-2 (K). The cells were processed for immunofluorescence using an antibody against LAMP1. Depletion of BICD2 results in perinuclear clustering of lysosomes. (L) The number of motile lysosome particles was quantified in HeLa cells transfected with either a control siRNA or an siRNA targeting BICD2. The number of motile lysosomes was greatly reduced in BICD2-depleted cells. An unpaired t test was used for this analysis . ****, p<0001. (M, M’) Cos7 cells were transfected with a plasmid encoding BICD2-mNeongreen. The cells were fixed and immunostained using an antibody against LAMP1. Overexpression of BICD2 results in the outward spreading of LAMP1 vesicles. The outline of the transfected cell is indicated in E’. The scale bar in the microscopy images is 20 µm.
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Figure 3—figure supplement 1—source data 1
Original files for western blots displayed in Figure 3—figure supplement 1A, B and C.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
PDF file for western blots displayed in Figure 3—figure supplement 1A, B and C with the bands marked.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig3-figsupp1-data2-v1.zip
Figure 3—video 1
Live imaging of SiR lysosome-labeled vesicles in HeLa cell transfected with a control siRNA.
Figure 3—video 2
Live imaging of SiR lysosome-labeled vesicles in HeLa cell transfected with an siRNA targeting BICD2.
Figure 4 with 1 supplement
BICD2 cargo binding domain mutants hyperactivate dynein.
(A) A co-immunoprecipitation experiment was performed using HEK cells expressing the indicated constructs. The tagged proteins were purified using V5 trap beads, and the co-precipitating proteins were analyzed by western blotting with the indicated antibodies. A greater amount of DIC and DCTN1 co-purified with mutant BICD2 compared to the wild-type protein. (B–E) HeLa cells expressing BICD2_wt (B), BICD2_N188T (C), BICD2_R694C (D), or BICD2_R747C (E) were fixed and processed for immunofluorescence using antibodies against V5 (cyan) and pericentrin (magenta). Merged images are also shown. All three mutants displayed a centrosomal localization pattern to varying degrees. The scale bar is 20 µm. (F) The centrosomal enrichment of BICD2_wt or mutant was quantified. (G) Schematic of the peroxisome tethering assay. (H) The average distance of peroxisomes to the nucleus was calculated on a cell-by-cell basis. In comparison to wild-type BICD2, all three mutants showed increased clustering of peroxisomes close to the nucleus. A one-way ANOVA was used for the quantifications shown in panels F and H with the values compared to the mean of BICD2_wt. n=not significant, **, p<0.01, ***, p<0.001, ****, p<0001.
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Figure 4—source data 1
Original files for western blots displayed in Figure 4A.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig4-data1-v1.zip
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Figure 4—source data 2
PDF file for western blots displayed in Figure 4A with the bands marked.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig4-data2-v1.zip
Figure 4—figure supplement 1
BICD2 localization and interaction with dynein and KIF5B.
(A B) Quantification of binding of BICD2_wt and mutants with DIC (A) and DCTN1 (B). The level of binding for the mutants was compared to BICD2_wt. A one-way ANOVA was used for this analysis. ns = not significant, *, p≤0.05. (C) A representative image of HeLa cells expressing BICD2-wt_TurboID. The cells were fixed and processed using antibodies against V5 (which recognizes TurboID, green, C) and GM130 (a Golgi marker, red, C’). The cells were also counterstained with DAPI (cyan, merged image in C’’). Wild-type BICD2_mTurboID partially co-localized with the centrosome. (D–G) Representative images of HEK cells expressing mGreenLantern-positive peroxisomes and co-expressing BICD2_wt (A), BICD2_N188T (B), BICD2_R694C (C), and BICD2_R747C (D) fused to the FKBP dimerization domain. The addition of rapamycin causes the tethering of peroxisomes to the indicated BICD2 construct. BICD2 mutants result in greater perinuclear clustering of peroxisome in comparison to the wild-type protein. (H) Lysates were prepared from cells expressing the indicated constructs and biotinylated proteins were purified using streptavidin beads. The purified proteins were analyzed by western blotting using an antibody against KIF5B. (I) The binding results in panel H were quantified. The level of binding for the mutants was compared to BICD2_wt. A one-way ANOVA was used for this analysis. ns = not significant, *, p≤0.05, ***, p<0.001. BICD2_R694C and BICD2_R747C displayed reduced binding to KIF5B. The scale bar is 20 μm.
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Figure 4—figure supplement 1—source data 1
Original files for the western blot shown in Figure 4—figure supplement 1H.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Original files for the western blot shown in Figure 4—figure supplement 1H with bands marked.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig4-figsupp1-data2-v1.zip
Figure 5
Localization of BICD2 wild-type and mutants in neurons.
(A–D) E18 rat hippocampal neurons were transfected with the indicated constructs. Two days after transfection, the cells were fixed and processed for immunofluorescence using a V5 antibody. The axon outline for cells expressing BICD2_R694C and BICD2_R747C is indicated. Signal for wild-type BICD2 could be detected in the cell body and axon. A similar phenotype was noted for BICD2_N188T. By contrast, BICD2_R694C and BICD2_R747C displayed reduced axonal signal. The scale bar is 100 µm. The signal for BICD2 is displayed using the ‘red hot’ LUT in FIJI. (E) Quantification of the cell body enrichment of BICD2 wild-type and mutant. (F) The axon length of neurons expressing either wild-type or mutant alleles of BICD2 was quantified. Expression of BICD2 mutants correlated with shorter axonal lengths. A one-way ANOVA was used for the quantifications shown in panels E and F, and the values were compared to the mean of BICD2_wt. n=not significant, **, p<0.01, ***, p<0.001, ****, p<0.0001.
Figure 6 with 1 supplement
BICD2 mutations are associated with altered interactomes.
(A–C). Volcano plots comparing the interactome of BICD2_wt vs BICD2_N188T (A), vs BICD2_R694C (B) and vs BICD2_R747C (C). Interacting proteins that show at least a twofold change in comparison to BICD2_wt and have a p value of at least 0.05 are indicated in the shaded boxes. Red boxes indicate proteins that display a greater interaction with BICD2 mutants vs the wild-type, whereas blue boxes indicate proteins that display a lower interaction vs the wild-type protein. (D) The proteomics results were validated by repeating the experiment and analyzing the bound fractions using the indicated antibodies. Streptavidin beads were used to purify the biotinylated proteins. (E–G) Quantification of binding of BICD2_wt and mutants with RANBP2 (E), VPS41 (F), and CSPP1 (G). The level of binding for the mutants was compared to BICD2_wt. Consistent with the proteomics results, BICD2_R747C displayed reduced binding to RANBP2 and VPS41. All three mutants bound CSPP1 at a greater level than the wild-type. A one-way ANOVA was used for this analysis. ns = not significant, *, p≤0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001.
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Figure 6—source data 1
Original files for western blots displayed in Figure 6D.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig6-data1-v1.zip
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Figure 6—source data 2
PDF file for western blots displayed in Figure 6D with the bands marked.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig6-data2-v1.zip
Figure 6—figure supplement 1
BICD2 interaction with nuclear import receptors and the HOPS complex.
(A, B) HEK cells expressing the indicated constructs were used in a binding experiment. Biotinylated proteins were purified using streptavidin beads. Bound proteins were eluted and analyzed by blotting using the indicated antibodies. Importin beta is biotinylated by wild-type BICD2 and BICD2_R694C. However, the binding between the two proteins is reduced in the BICD2_R747C background. Two replicate experiments are shown. (C) HEK cells expressing BICD2_wt were transfected with the indicated siRNA. Three days after the siRNA transfection, lysates were prepared and biotinylated proteins were purified using streptavidin beads. Bound proteins were analyzed by blotting using the indicated antibodies. The ability of Importin beta to be biotinylated by BICD2 depends on RANBP2. (D) Lysates were prepared from HEK cells expressing the indicated constructs. Binding was performed using streptavidin beads and the bound proteins were analyzed by blotting. VPS16 and VPS18 displayed reduced binding to BICD2_R747C.
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Figure 6—figure supplement 1—source data 1
Original files for western blots shown in Figure 6—figure supplement 1A-D.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Original files for western blots shown in Figure 6—figure supplement 1A-D with bands indicated.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig6-figsupp1-data2-v1.zip
Figure 7 with 1 supplement
BICD2_R747C is associated with a gain-of-function interaction with GRAMD1A.
(A) Lysates from cells expressing BICD2_wt and mutants were incubated with streptavidin beads to purify biotinylated proteins. Bound proteins were eluted and analyzed by blotting using the indicated antibodies. BICD2_R747C interacted with substantially more GRAMD1A than either the control, BICD2_wt, or the other mutants. (B) Quantification of binding of BICD2_wt and mutants with GRAMD1A. The level of binding for the mutants was normalized to BICD2_wt. A one-way ANOVA was used for this analysis. ns = not significant, ***, p<0.001. (C–F) Cos7 cells were co-transfected with constructs expressing either BICD2_wt or mutant (magenta) along with a plasmid expressing GRAMD1A-mScarlet3 (cyan). Except for cells expressing BICD2_R747C, GRAMD1A was localized to the ER. By contrast, GRAMD1A was highly enriched at the centrosome in cells expressing BICD2_R747C. The scale bar is 20 μm. (G) Quantification of the co-localization between BICD2_wt and mutants with GRAMD1A. A one-way ANOVA was used for this analysis and the values were compared to the mean of BICD2_wt. ns = not significant, ****, p<0.0001.
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Figure 7—source data 1
Original files for western blots displayed in Figure 7A .
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig7-data1-v1.zip
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Figure 7—source data 2
PDF file for western blots displayed in Figure 7A with the bands marked.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig7-data2-v1.zip
Figure 7—figure supplement 1
BICD2 interaction and co-localization with GRAMD1A.
(A) HEK cells expressing the indicated constructs were transfected with an siRNA targeting endogenous BICD2. Three days after transfection, lysates were prepared, and biotinylated proteins were purified using streptavidin beads. The precipitated proteins were analyzed by blotting using antibodies against RANBP2 and GRAMD1A. Whereas the interaction between BICD2_R747C and RANBP2 is reduced in cells depleted of endogenous BICD2, the interaction of this mutant with GRAMD1A is increased in these same cells. (B-F) Cos7 cells were co-transfected with plasmids encoding BICD2_R747C-mNeon (green, B, C) and GRAMD1A-mScarlet3 (cyan, D). The cells were fixed and processed using an antibody against Pericentrin (a centrosome marker, magenta, E). The merged image is shown in (F). (G, H) Cos7 cells were co-transfected with plasmids encoding an ER marker (Addgene plasmid #137805, Chertkova et al., 2020) and BICD2_wt (G, G’) or BICD2_R747C (H, H’). In contrast to what was observed with GRAMD1A, expression of BICD2_R747C does not alter the localization of the ER marker. The scale bar is 20 μm.
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Figure 7—figure supplement 1—source data 1
Original data for western blots shown in Figure 7—figure supplement 1A.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig7-figsupp1-data1-v1.zip
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Figure 7—figure supplement 1—source data 2
Original data for western blots shown in Figure 7—figure supplement 1A with bands indicated.
- https://cdn.elifesciences.org/articles/107503/elife-107503-fig7-figsupp1-data2-v1.zip
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HeLa | ATCC | HeLa (ATCC CCL-2) | |
| Cell line (Cercopithecus aethiops) | COS-7 | ATCC | CRL-1651 | |
| Cell line (H. sapiens) | Flp-In–293 Cell Line | Thermo Fisher | R75007 | |
| Biological sample (Rattus norvegicus) | E18 Rat hippocampus | Transnetyx | KTSDEHP | |
| Antibody | Anti-V5 (mouse monoclonal) | Invitrogen | R960-25 | 1:10,000 for western; 1:1000 for immunofluorescence |
| Antibody | Anti-VPS41 (rabbit polyclonal) | Abcam | ab181078 | 1:1000 for western |
| Antibody | Anti-VPS16 (rabbit polyclonal) | Proteintech | 17776–1-AP | 1:1000 for western |
| Antibody | Anti-VPS18 (rabbit polyclonal) | Proteintech | 67590–1-Ig | 1:5000 for western |
| Antibody | Anti-RANBP2 (mouse monoclonal) | Santa Cruz | sc-74518 | 1:400 for western |
| Antibody | Anti-LAMP1 (rabbit polyclonal) | Cell Signaling Technology | 9091T | 1:600 for immunofluorescence |
| Antibody | Anti-DCTN1 (rabbit polyclonal) | ThermoFisher | PA5-21289 | 1:2000 for western |
| Antibody | Anti-DIC (mouse monoclonal) | Millipore Sigma | D5167 | 1:1000 for western |
| Antibody | Anti-Pericentrin (rabbit polyclonal) | Abcam | ab4448 | 1:200 for immunofluorescence |
| Antibody | Anti-CSPP1 (rabbit polyclonal) | Proteintech | 11931–1-AP | 1:500 for western |
| Antibody | Anti-GRAMD1A (rabbit polyclonal) | Novus Biologicals | NBP2-32148 | 1:1000 for western |
| Antibody | Anti-alpha Tubulin (mouse polyclonal) | Millipore Sigma | T6199 | 1:50,000 for western |
| Antibody | Anti-Dynein heavy chain (mouse monoclonal) | Santa Cruz | sc-514579 | 1:1000 for western |
| Antibody | Anti-KIF5B (rabbit polyclonal) | Proteintech | 21632–1-AP | 1:4000 for western |
| Antibody | Anti-BICD2 (mouse monoclonal) | Thermo Fisher | MA5-23522 | 1:1000 for western |
| Antibody | Anti-Importin beta (rabbit polyclonal) | Cell Signaling Technologies | 60769 | 1:1000 for western |
| Antibody | Anti-GM130 (rabbit polyclonal) | Cell Signaling Technologies | 12480 | 1:1250 for immunofluorescence |
| Antibody | Anti-Cyclin B (rabbit polyclonal) | Cell Signaling Technologies | 4138T | 1:200 for immunofluorescence |
| Antibody | Goat-anti mouse Alexa488 | Thermo Fisher | A-11029 | 1:400 for immunofluorescence |
| Antibody | Goat-anti rabbit Alexa488 | Thermo Fisher | A-11034 | 1:400 for immunofluorescence |
| Antibody | Goat-anti mouse Alexa555 | Thermo Fisher | A-21424 | 1:400 for immunofluorescence |
| Antibody | Goat-anti rabbit Alexa555 | Thermo Fisher | A-21428 | 1:400 for immunofluorescence |
| Antibody | Goat-anti mouse HRP | Thermo Fisher | 31430 | 1:5000 for western |
| Antibody | Goat-anti rabbit HRP | Thermo Fisher | 31460 | 1:5000 for western |
| Recombinant DNA reagent | pCDNA5/FRT/TO | Thermo Fisher | V652020 | |
| Recombinant DNA reagent | mRFP-mTrbo cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_wt-mTrbo cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_delCC3-mTrbo cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_delCC1-mTrbo cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_CC3-mTrbo cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_N188T-mTrbo cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_R694C-mTrbo cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_R747C-mTrbo cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | GFP-VPS41 | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | pEGFP-C3 | Clontech | Discontinued by manufacturer | |
| Recombinant DNA reagent | pLVX-Puro | Takara | 631849 | |
| Recombinant DNA reagent | BICD2_wt-mNeon in pLVX-Puro | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_N188T-mNeon in pLVX-Puro | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_R694C-mNeon in pLVX-Puro | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_R747C-mNeon in pLVX-Puro | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | PEX3-mGreenLantern-FKBP in pLVX-Puro | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_wt-FRB cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_N188T-FRB cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_R694C-FRB cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | BICD2_R747C-FRB cloned into pCDNA5/FRT/TO | This paper | Available upon request | Materials and methods |
| Recombinant DNA reagent | EGFP-RAB6AQ72L | Addgene | Plasmid #49483 | |
| Recombinant DNA reagent | ER-mScarletI | Addgene | Plasmid #137805 | |
| Recombinant DNA reagent | GFP-BICD2 | Addgene | Plasmid #164626 | |
| Sequence-based reagent | control siRNA | Thermo Fisher | 4390843 | |
| Sequence-based reagent | DYNC1H1 siRNA | Thermo Fisher | 4390824 | Assay ID# s4200 |
| Sequence-based reagent | BICD2 siRNA-1 | Thermo Fisher | 4392420 | Assay ID# s23497 |
| Sequence-based reagent | BICD2 siRNA-2 | Thermo Fisher | 4392420 | Assay ID# s225943 |
| Sequence-based reagent | KIF5B siRNA | Thermo Fisher | 4390824 | Assay ID# s732 |
| Sequence-based reagent | RANBP2 siRNA | Thermo Fisher | 4390824 | Assay ID# s11775 |
| Sequence-based reagent | VPS41 siRNA | Thermo Fisher | 4392420 | Assay ID# s25770 |
| Commercial assay or kit | Streptavidin magnetic beads | Thermo Fisher | 88816 | |
| Commercial assay or kit | Streptavidin agarose beads | Thermo Fisher | 20357 | |
| Commercial assay or kit | V5 trap beads | Chromotek | v5ta-20 | |
| Commercial assay or kit | SiR lysosome kit | Cytoskeleton, Inc. | CY-SC012 | |
| Chemical compound, drug | D-biotin | Thermo Fisher | Β1595 | |
| Software, algorithm | Imaris 10.1 | Oxford Instruments | RRID:SCR_007370 | |
| Software, algorithm | Prism 10 | GraphPad | RRID:SCR_002798 | Version 10.6.1 |
| Software, algorithm | Fiji | ImageJ 1.54 p | RRID:SCR_002285 |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/107503/elife-107503-mdarchecklist1-v1.docx
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Supplementary file 1
The wild-type BICD2 interactome.
- https://cdn.elifesciences.org/articles/107503/elife-107503-supp1-v1.xlsx
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Supplementary file 2
The interactome of BICD2_N188T vs BICD2_wt.
- https://cdn.elifesciences.org/articles/107503/elife-107503-supp2-v1.xlsx
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Supplementary file 3
The interactome of BICD2_R694C vs BICD2_wt.
- https://cdn.elifesciences.org/articles/107503/elife-107503-supp3-v1.xlsx
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Supplementary file 4
The interactome of BICD2_R747C vs BICD2_wt.
- https://cdn.elifesciences.org/articles/107503/elife-107503-supp4-v1.xlsx
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Dominant spinal muscular atrophy linked mutations in the cargo binding domain of BICD2 result in altered interactomes and dynein hyperactivity
eLife 14:RP107503.
https://doi.org/10.7554/eLife.107503.3
