Single-molecule imaging of cytoplasmic dyneinin celluloreveals the mechanism of motor activation and cargo movement

Abstract

Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells (1). Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin and a cargo adaptor (2-5). Thereupon, dynein transports cargo towards the minus ends of microtubules. How this process of motor activation occurs is unclear, since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualise and track fluorescently tagged dynein. First, we observed that dynein that bound to the microtubule engaged in minus end-directed movement only ~30% of the time and resided on the microtubule for a short duration. Next, using high-resolution imaging in live and fixed cells, we discovered that dynactin remained persistently attached to microtubules, and endosomal cargo remained in proximity to the microtubules and dynactin. Finally, we employed two-colour imaging to visualise cargo movement effected by single motor binding. Taken together, we discovered a search strategy that is facilitated by dynein's frequent microtubule binding-unbinding kinetics: (1) in a futile event when dynein does not encounter cargo anchored in proximity to the microtubule, dynein unbinds and diffuses into the cytoplasm, (2) when dynein encounters cargo and dynactin upon microtubule binding, it moves cargo in a short run. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on reduction of dimensionality to enable minus end-directed transport in vivo.

HEATR5B associates with dynein-dynactin and selectively promotes motility of AP1-bound endosomal membranes

“…During several minutes of filming, the vast majority of the dual-labelled punctate structures exhibited short, oscillatory movements (Movie S1). This behaviour is reminiscent of the behaviour of early endosomal cargoes for microtubule motors in HeLa cells, which can take tens of minutes to traverse the cytoplasm (Flores-Rodriguez et al, 2011;Zajac et al, 2013;Tirumala et al, 2022). However, a small fraction of AP1σ1 puncta exhibited unidirectional movements of between 1.5 and 10 μm both towards and away from the perinuclear region with a mean instantaneous velocity of 260 ± 4 nm/s (± SEM; 22 particles) (Figure 3D and Movie S2).…”

HEATR5B associates with dynein‐dynactin and promotes motility of AP1‐bound endosomal membranes

“…During several minutes of filming, the vast majority of the dual‐labelled punctate structures exhibited short, oscillatory movements (Movie EV1). This behaviour is reminiscent of the behaviour of early endosomal cargoes for microtubule motors in HeLa cells, which can take tens of minutes to traverse the cytoplasm (Flores‐Rodriguez et al , 2011; Zajac et al , 2013; preprint: Tirumala et al , 2022). However, a small fraction of AP1σ1 puncta exhibited unidirectional movements of between 1.5 and 10 μm both towards and away from the perinuclear region with a mean instantaneous velocity of 260 ± 4 nm/s (± SEM; 22 particles) (Fig 3D; Movie EV2).…”

Cargo specificity, regulation, and therapeutic potential of cytoplasmic dynein

“…HILO microscopy generates a thin excitation plane of several microns 36 , increasing the signal/background ratio and decreasing photobleaching compared with conventional confocal microscopy because of the nonfocused illumination, thereby providing better spatial and temporal resolution and enabling near-single-molecule tracking of the motor protein inside living cells 37 . When the movement of mouse dynein heavy chain labeled with GFP in HeLa cells was tracked via HILO microscopy, approximately 30% of the dynein bound to MTs showed processive movements toward the minus end of the MT over a relatively short period (~0.5 s), and the average length was less than one micron 38 . This finding implies that when traveling distances greater than one micron, dynein-dynactin-cargo adaptor complexes consistently undergo binding and unbinding.…”