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.

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

“…Purity of both complexes was demonstrated by SDS–PAGE, and the subunit composition of the dynactin complex was verified by mass spectrometry (Fig EV1A). To be able to study simultaneously dynamic microtubule end tracking and processive motility initiation of dynein/dynactin, we adjusted the conditions of previous in vitro reconstitution experiments, using here a buffer that was intermediate in ionic strength compared to previous dynein motility studies on static microtubules (Trokter et al , 2012; McKenney et al , 2014; Schlager et al , 2014) and microtubule end tracking experiments (Duellberg et al , 2014; Roberts et al , 2014) (Materials and Methods). …”

“…The interaction between dynein and dynactin is stabilised by a variety of cargo adaptors, such as Bicaudal‐D2 (BicD2), which activates processive dynein motion (McKenney et al , 2014; Schlager et al , 2014; Hoogenraad & Akhmanova, 2016). It is not known how this stabilisation and activation of processive motion influences the end tracking behaviour of dynein.…”

“…Despite being the key dynein regulator, dynactin itself interacts only weakly with dynein (McKenney et al , 2014; Schlager et al , 2014). Additional adaptor proteins that recruit dynein to cargoes stabilise this interaction, leading to ternary complex formation and activation of processive dynein motility (McKenney et al , 2014; Schlager et al , 2014).…”

“…Despite being the key dynein regulator, dynactin itself interacts only weakly with dynein (McKenney et al , 2014; Schlager et al , 2014). Additional adaptor proteins that recruit dynein to cargoes stabilise this interaction, leading to ternary complex formation and activation of processive dynein motility (McKenney et al , 2014; Schlager et al , 2014). Ternary complex formation is thought to release dynein from its autoinhibited state, possibly by separating the two motor domains (Chowdhury et al , 2015; Urnavicius et al , 2015; Carter et al , 2016).…”

“…One example is the metazoan‐specific adapter protein Bicaudal‐D2 (BicD2) that is critical for bidirectional transport of mRNA particles and contributes to the positioning of the endoplasmic reticulum, the Golgi apparatus and the nucleus (Bullock & Ish‐Horowicz, 2001; Hoogenraad et al , 2001; Hoogenraad & Akhmanova, 2016). The N‐terminal coiled‐coil of BicD2 mediates the interaction between the dynein tail and dynactin, which is crucial for processive minus‐end‐directed dynein motion (McKenney et al , 2014; Schlager et al , 2014; Chowdhury et al , 2015; Urnavicius et al , 2015), whereas the C‐terminal part of BicD2 interacts with Rab receptors on cargo membranes (Hoogenraad et al , 2001; Hoogenraad & Akhmanova, 2016). …”