Processive bidirectional motion of dynein–dynactin complexes in vitro

Abstract

Cytoplasmic dynein is the primary molecular motor responsible for transport of vesicles, organelles, proteins and RNA cargoes from the periphery of the cell towards the nucleus along the microtubule cytoskeleton of eukaryotic cells. Dynactin, a large multi-subunit activator of dynein, docks cargo to the motor and may enhance dynein processivity. Here, we show that individual fluorescently labelled dynein-dynactin complexes exhibit bidirectional and processive motility towards both the plus and minus ends of microtubules. The dependence of this activity on substrate ATP concentration, nucleotide analogues and inhibitors suggests that bidirectional motility is an active energy-transduction property of dynein-dynactin motor mechano-chemistry. The unique motility characteristics observed may reflect the flexibility of the dynein structure that leads to an enhanced ability to navigate around obstacles in the cell.

In vitro reconstitution of a highly processive recombinant human dynein complex

“…The motile behaviour of mammalian dynein has been studied using complexes purified from brain (Mallik et al , 2005; Ross et al , 2006; Miura et al , 2010; Ori‐McKenney et al , 2010; Walter et al , 2010) and tissue culture cells (Ichikawa et al , 2011), as well as complexes reconstituted from individual, recombinant components (Trokter et al , 2012). Movement of individual mammalian dynein complexes has been assayed by adhering the motor to beads (King & Schroer, 2000; Mallik et al , 2005; Walter et al , 2010), labelling accessory proteins (Ross et al , 2006; Miura et al , 2010) or by GFP tagging of the motor (Trokter et al , 2012). The extent to which individual dynein complexes can take multiple successive steps without detaching from the microtubule, a behaviour termed processivity, varied in these studies.…”

“…The extent to which individual dynein complexes can take multiple successive steps without detaching from the microtubule, a behaviour termed processivity, varied in these studies. Some groups reported a subset of dyneins undergoing processive movements with an average run length of approximately 0.7–1 μm (King & Schroer, 2000; Mallik et al , 2005; Culver‐Hanlon et al , 2006; Ross et al , 2006), whereas others documented substantially shorter excursions (Ori‐McKenney et al , 2010). Other studies reported no measurably processive movement (Miura et al , 2010; Trokter et al , 2012).…”

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

“…The critical dynactin subunit for plus‐end tracking is p150 Glued (called p150 here). Its N‐terminal CAP‐Gly domain protrudes from the shoulder of the dynactin complex (Chowdhury et al , 2015; Urnavicius et al , 2015) and binds directly to microtubules (Culver‐Hanlon et al , 2006; Peris et al , 1998; Ross et al , 2006), as well as to EBs (Honnappa et al , 2006). In vitro reconstitution experiments with a p150 fragment showed that the p150 CAP‐Gly domain and the first coiled‐coil of p150, which interacts with the intermediate chain of the dynein complex (Karki & Holzbaur, 1995; Vaughan & Vallee, 1995; King et al , 2003), were sufficient for mediating EB‐dependent end tracking of the human dynein complex (Duellberg et al , 2014).…”

From the Cell Membrane to the Nucleus: Unearthing Transport Mechanisms for Dynein

“…In experiments on dynein alone, is seen to move progressively, however it is a simplification of what is seen in experiments on the dynein moving cargo. Experiments do show cargos within a cell being moved in the direction of the nucleus over time, however there are also pauses in movement and the cargo can travel backwards or move in a direction which is not solely radial [21,12,42,46]. Currently, the available experimental data does not show how the dynein itself is behaving at these times, as we only see the position of the endosome.…”

“…Currently, the available experimental data does not show how the dynein itself is behaving at these times, as we only see the position of the endosome. The pauses and retrograde transport may be a result of the action of kinesins on the cargo, either in a tug-of-war scenario, dynein may occasionally fall off a microtubule, or simply become inhibited by a crowded cytoplasm [1,21,38,42,48]. In addition, non-radial movement, could also be a result of dynein being detached from the cargo or the microtubule.…”