In vitro reconstitution of a highly processive recombinant human dynein complex

Abstract

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.

Keywords: Bicaudal‐D; dynactin; dynein; microtubules; processivity.

Figure 1. Expression and purification of complete recombinant human dynein complexes from a single baculovirus

1. Schematic overview of the dynein genes present in the pDyn1 and pDyn2 plasmids and the assembly of pDyn3 using Cre recombinase. pDyn3 was subsequently integrated into the baculoviral genome by Tn7 transposition to form DynBac. T indicates a Tobacco Etch Virus (TEV) protease cleavage site; black triangles and black rectangles represent PolH promoter and SV40 terminator sequences, respectively. Not to scale.

2. Coomassie-stained SDS–PAGE gel of purified recombinant dynein complex. Inset is the 10–15 kDa range from a gel with better low-molecular-weight separation on which bands corresponding to the different light chains can be discriminated.

3. SEC-MALS of recombinant dynein. Mean observed molar mass (Obs.) and expected (Exp) molar mass are indicated. Expected molar mass was calculated for a dimeric complex of the DHC, DIC, DLIC, Tctex, Robl and LC8 chains. V₀ indicates the void volume of the column.

Source data are available online for this figure.

Figure 2. Structural comparison of recombinant human and endogenous pig dynein

Representative negative stain EM 2D class averages. Particles were aligned on the tail region and sub-classified based on the degree of inter-head separation (see Materials and Methods and Supplementary Fig S2 for details). The recombinant human dynein (bottom row) is structurally similar to dynein purified from pig brains (top row). Left-hand images show phi-particle arrangement (Amos, 1989). Three distinct tail domains are numbered (see text for details). Scale bar, 20 nm.

Figure 3. BICD2N and dynactin are sufficient to convert dynein into a highly processive motor

A Stills from a microtubule gliding assay with immobilised recombinant human dynein. Microtubules move with their plus ends leading (plus ends (coloured arrowheads) are labelled with greater incorporation of HiLyte-488 tubulin). t, time. The mean gliding speed per microtubule was 0.30 ± 0.11 μm/s (n = 85 microtubules) in 30 mM HEPES/KOH, 5 mM MgSO₄, 1 mM DTT, 1 mM EGTA, 40 μM taxol, 1 mg/ml α-casein, 2.5 mM ATP, pH 7.0 and 0.48 ± 0.06 μm/s (n = 112 microtubules) in the same buffer with the addition of 50 mM KCl. The latter mean velocity is similar to that reported by Trokter et al (2012) for gliding assays with their recombinant human dynein, which used a similar salt concentration in the buffer. These stills are from an experiment with low-salt buffer. B Representative kymographs of TMR–dynein motility in the presence and absence of BICD2N and dynactin. Blue, red and yellow arrowheads show examples of static, diffusive and highly processive TMR–dynein complexes, respectively. − and + indicate polarity of microtubule ends. C Quantification of proportion of TMR–dyneins that exhibit static, diffusive and processive (unidirectional, minus end-directed) behaviour with the indicated experimental conditions. Dynactin was added in a twofold excess to dynein, except in one condition when it was in an 80-fold excess. Mean (± SEM) values per chamber are shown (derived from 3 to 5 chambers for each condition). For each condition, between 200 and 300 complexes were analysed in total. ***P < 0.001 (two-tailed t-test) compared to TMR–dynein alone (no parentheses) or to TMR–dynein + dynactin + BICD2N in the absence of vanadate (parentheses). D, E Distribution of mean velocity (D) and mean run length (E) of processive (unidirectional, minus end-directed) bouts of motion. A run was defined as a bout of TMR–dynein motion that could be terminated by a pause or detachment from the microtubule. Some processive runs contained switches between bouts of motion with different constant velocities. Mean velocity was therefore calculated from these constant velocity segments. Data information: In all experiments, ATP concentration was 2.5 mM ATP (vanadate experiments included 100 μm vanadate and 2.5 mM ATP). Microtubules were stabilised with GmpCpp.

Figure 4. BICD2N and dynactin can induce robust processivity by regulating individual dynein complexes

A Cartoon exemplifying how a mixture of dynein labelled with different fluorophores can provide insights into how BICD2N and dynactin affect the oligomeric status of the dynein complex. In the idealised example shown, an exactly 50:50 mixture of TMR–dynein and Alexa647(A647)–dynein is predicted to result in a 25:25:50 proportion of dyneins with, respectively, signals from TMR only, A647 only and both fluorophores if BICD2N and dynactin induce dimerisation of dynein complexes. Induction of higher order oligomers is predicted to result in a greater proportion of dual-labelled puncta on microtubules. B, C Kymograph and quantification of mean proportion of microtubule-associated dynein puncta that have signals from TMR only, A647 only and both fluorophores when TMR–dynein and A647–dynein are mixed in the absence (B) and presence (C) of BICD2N and dynactin. Contrast of images was enhanced so that any puncta containing both dyes could be visualised readily. Example of a dual colour (white) punctum is labelled with a yellow arrowhead. Note that slightly more dynein puncta are labelled with TMR than A647, presumably as a result of multiple manual handling steps in the procedure (Supplementary Fig S5). Mean values per chamber are shown, with 6 chambers from 2 independent dynein, BICD2N and dynactin preparations analysed (10–20 kymographs analysed per chamber for each condition). D Quantification of mean fluorescence intensity of TMR signals from puncta of TMR–dynein that display static, diffusive and processive movements in the absence and presence of dynactin and BICD2N. Mean values per chamber are displayed, with four chambers each for dynein and for dynein + dynactin + BICD2N (error bars show SEM). See Supplementary Fig S6 for distribution and mean fluorescence intensity of individual particles. Mean fluorescence intensity of processive TMR–dyneins in the absence of dynactin and BICD2N could not be accurately determined due to their rarity.

Figure 5. Dynein, dynactin and BICDN form a complex, with dynein and dynactin interacting in a well-ordered structure

1. Size-exclusion chromatography traces for a mixture of dynein and dynactin alone (black trace; 1 dynein complex to 2 dynactin complexes) and dynein, dynactin and BICDN (red trace; 1 dynein complex to 2 dynactin complexes to 20 BICD2N dimers). DDB, dynein–dynactin–BICD2N complex. V₀ indicates the void volume of the column.

2. SYPRO Ruby-stained SDS–PAGE gel of the pooled and concentrated fractions collected from the DDB peak in (A). In addition to dynein subunits and BICD2N, multiple bands corresponding to dynactin subunits are observed. p135 is an spliceoform of p150 (Tokito et al, 1996). Note that BICD2N has a predicted molecular mass of 72.4 kDa due to the presence of the GFP tag.

3. Representative negative stain EM single particles (low-pass filtered to 30 Å) of the DDB complex and recombinant human dynein. Note the significantly larger tail domain of the DDB complex (white bracket) and the range of head positions for both complexes. Scale bar, 20 nm.

4. 2D class average of the DDB tail compared to 2D class averages of dynactin and the recombinant human dynein tail. Alignment of the dynein and DDB tails was performed by applying a binary mask that excluded the flexible dynein heads to all particles (see Supplementary Fig S2 and Materials and Methods). This procedure results in the head domains appearing as a blur following removal of the mask. Dynactin structural features are labelled as follows: p, pointed end; s, shoulder/projecting arm; b, barbed end. The dashed lines allow a size comparison of the DDB tail domain to the dynein tail and dynactin alone. Dynactin appears to be positioned approximately along the length of dynein tail domain in the DDB complex. The positions of the pointed end, shoulder/projecting arm and barbed end cannot be unambiguously determined in the class average of the DDB tail. Scale bar, 20 nm.

Source data are available online for this figure.