In vitro reconstitution of a highly processive recombinant human dynein complex

Cytoplasmic dynein is an approximately 1.4 MDa multi-protein complex that transports many cellular cargoes towards the minus ends of microtubules. Several in vitro studies of mammalian dynein have suggested that individual motors are not robustly processive, raising questions about how dynein-associated cargoes can move over long distances in cells. Here, we report the production of a fully recombinant human dynein complex from a single baculovirus in insect cells. Individual complexes very rarely show directional movement in vitro. However, addition of dynactin together with the N-terminal region of the cargo adaptor BICD2 (BICD2N) gives rise to unidirectional dynein movement over remarkably long distances. Single-molecule fluorescence microscopy provides evidence that BICD2N and dynactin stimulate processivity by regulating individual dynein complexes, rather than by promoting oligomerisation of the motor complex. Negative stain electron microscopy reveals the dynein–dynactin–BICD2N complex to be well ordered, with dynactin positioned approximately along the length of the dynein tail. Collectively, our results provide insight into a novel mechanism for coordinating cargo binding with long-distance motor movement.

4°C, with the supernatant (p2 virus) stored at 4°C in the dark. All Sf9 cells were cultured in serum free media (either Sf900-II (Life Technologies) or Insect Express (Lonza)).

Purification of native dynein and dynactin
Native dynein was purified from pig brains using microtubule affinity purification as described by Bingham et al. (Bingham et al, 1998). The large scale purification protocol of Bingham et al. (Bingham et al, 1998) was used to purify dynactin without a microtubule affinity purification step. For both complexes a final gel filtration step using a TSKgel G4000SW XL column with a TSKgel SW XL guard column (TOSOH Bioscience) equilibrated in GF150 buffer was added to the protocol. Typically two pig brains were used per dynactin preparation.

Production and purification of recombinant BICD2N
Purification was performed essentially as described for recombinant dynein (Materials and methods) with the following modifications. After TEV cleavage the beads were removed and the protein of interest concentrated to 3 -10 mg/ml and snap frozen in liquid nitrogen in 100 µl aliquots. TEV protease was removed from thawed aliquots as described for dynein using size-exclusion chromatography. Peak fractions were collected, pooled, concentrated to 0.5 -10 mg/ml using Amicon concentrators and snap frozen in liquid nitrogen in 3 -5 ul aliquots. All purification steps were performed at 4°C, with frozen proteins stored at -80°C.

Size-exclusion chromatography of DDB complexes
Purified recombinant dynein, pig dynactin and BICD2N were mixed at the required molar ratio and incubated on ice for 10 to 20 min directly followed by size-exclusion chromatography using a using a TSKgel G4000SW XL column with a TSKgel SW XL guard column equilibrated in GF150 buffer.

Size-exclusion chromatography-multi angle light scattering (SEC-MALS)
SEC-MALS employed a Heleos II 18 angle light scattering instrument (Wyatt) coupled to an Optilab refractive index detector (Wyatt). Samples were resolved on a TSKgel G4000SW XL column with a TSKgel SW XL guard column equilibrated in GF150 buffer before passing through the light scattering and refractive index detectors using a standard SEC-MALS format. The excess differential refractive index (based on 0.186 ΔRI for 1 g/ml) was used to determine protein concentration.
The concentration and observed scattered intensity were used to determine the molar mass from the intercept of the Debye plot (using Zimm's model in the ASTRA software package (Wyatt)). Measurements were calibrated using bovine serum albumin (Thermo Scientific). Images are from human recombinant dynein, pig brain dynein or from the size exclusion chromatography peak that contained a complex of human recombinant dynein, pig brain dynactin and mouse recombinant BICD2N (DDB; see Figure 5A  In order to allow tail features to be aligned the indicated binary masks were applied in RELION. In the absence of such a step, the alignment process was compromised by the high variability in dynein head positions within the population of individual particles. Prior to this step, 2D classification without a mask resulted into the native pig dynein and human recombinant dynein particles being grouped into phi particle and head-separated classes. These classes were aligned separately using the binary masks shown. The EM images shown are the result of applying the binary masks during further alignment of all the individual particles of each class. Heads appear as a blur due to their variable positions with respect to the tail in the population. D Two examples of kymographs from sequential dual colour imaging of BICD2N, which is tagged with GFP (see Results), and TMR-dynein in the presence of unlabelled dynactin. GFP-BICD2N signal is frequently detected on processive TMR-dyneins, but rarely on non-processive TMR-dyneins. One example of a processive TMR-dynein is shown per kymograph (arrowheads). Note that the low intensity and rapid photobleaching of the GFP signal makes accurate assessment of the degree of co-incidence of signals from BICD2N and dynein on processive and non-processive dyneins impossible. In order to better visualise GFP signal on microtubule-associated dynein above background levels, the concentration of GFP-BICD2N was halved compared to the assays in Figure 3B, C and S3A -C. Thus, a ratio of 10 BICD2N dimers: 1 dynein complex: 2 dynactin complexes was used.

Supplementary Figure Legends
Polarity of microtubule ends in A -D is indicated by -and +. Figure S4 -Coomassie stained SDS-PAGE gels of dynactin purified from pig brain and recombinant BICD2N purified from Sf9 cells.
The preparations have a high degree of purity. Note that the molecular mass of BICD2N is increased by the fusion to GFP.  A Trace for a mixture of dynein and BICD2N at a ratio of 1 dynein complex to 20 BICD2N dimers. No complex formation between dynein and BICD2N is observed.

B
Trace for a mixture of dynein and dynactin at a ratio of 1 dynein complex to 2 dynactin complexes. No complex formation between dynein and dynactin is observed. Note that the trace is the same as the black trace in Figure 5A.
C Trace for a mixture of dynein, dynactin and BICD2N at a ratio of 1 dynein complex to 2 dynactin complexes to 20 BICD2N dimers. A high molecular weight peak is observed that is indicative of formation of dynein-dynactin-BICD2N complexes. Note that the trace is the same as the red trace in Figure 5A. Coomassie stained SDS-PAGE gels of pooled and concentrated fractions of the associated size-exclusion chromatography peaks are also shown except for the DDB peak in C, for which the gel is shown in Figure 5B.