Dynactin functions as both a dynamic tether and brake during dynein-driven motility

Abstract

Dynactin is an essential co-factor for most cellular functions of the microtubule motor cytoplasmic dynein, but the mechanism by which dynactin activates dynein remains unclear. Here, we use single molecule approaches to investigate dynein activation by the dynactin subunit p150Glued. We investigate the formation and motility of a dynein-p150Glued co-complex using dual-color TIRF microscopy. p150Glued recruits and tethers dynein to the microtubule in a concentration-dependent manner. Single molecule imaging of motility in cell extracts demonstrates that the CAP-Gly domain of p150Glued decreases the detachment rate of the dynein-dynactin complex from the microtubule and also acts as a brake to slow the dynein motor. Consistent with this important role, two neurodegenerative disease-causing mutations in the CAP-Gly domain abrogate these functions in our assays. Together, these observations support a model in which dynactin enhances the initial recruitment of dynein onto microtubules and promotes the sustained engagement of dynein with its cytoskeletal track.

Influence of Fluorescent Tag on the Motility Properties of Kinesin-1 in Single-Molecule Assays

“…6). The HALO tag and TMR ligand were recently used to label dynactin subunits expressed in COS7 cells for single-molecule analysis of dynein motility (32). We also found that kinesin-1 motors tagged with SNAP-TMR and HALO-TMR showed different run lengths in single-molecule motility assays (Fig.…”

Stress-Induced CDK5 Activation Disrupts Axonal Transport via Lis1/Ndel1/Dynein

“…For a 70–80% confluent 10 cm plate, cells were lysed in 100 µl lysis buffer, which was then diluted 1:20 or 1:50 for single molecule imaging. Anti-tubulin antibody (Sigma) was incubated in flow chambers constructed as described (Ayloo et al, 2014) for 5 minutes, followed by blocking with 5% pluronic F-127 (Sigma) for 5 minutes. Rhodamine-labeled taxol-stabilized microtubules were flowed into the chamber and bound to the anti-tubulin antibody for 10 minutes.…”

Axonal Transport: Cargo-Specific Mechanisms of Motility and Regulation

“…This subunit binds directly to dynein intermediate chain (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995), and also binds directly to microtubules via a CAP-Gly (Cytoskeletal Associated Protein-Glycine-rich) domain (Waterman-Storer et al, 1995) and a lower affinity basic domain found in neuronal isoforms of p150 Glued (Culver-Hanlon et al, 2006; Dixit et al, 2008a). In vitro assays demonstrate that these independent microtubule-binding domains increase the processivity of the dynein-dynactin motor complex (King and Schroer, 2000; Ross et al, 2006) by enhancing the association of the motor with the microtubule (Ayloo et al, 2014). In neurons, the CAP-Gly domain of dynactin has a key role in the initiation of retrograde transport in the distal axon (Lloyd et al, 2012; Moughamian and Holzbaur, 2012).…”